Compositions, combinations and related methods for photoimmunotherapy

ABSTRACT

Provided herein are conjugates, compositions and methods for use in photoimmunotherapy, such as photoimmunotherapy induced by activation of a phthalocyanine dye conjugated to a targeting molecule that binds a protein on cell, for example, an IR700-antibody conjugate. In some embodiments, the phthalocyanine-dye conjugate can be activated by irradiation with near-infrared light. Features of the conjugates, compositions and methods, including the dose of the conjugate, provide various advantages, such as lower toxicity and/or improved efficacy. In some embodiments, also provided is a dual label phthalocyanine-dye conjugate in which the targeting molecule is conjugated to an additional fluorescent dye, which can be used for photoimmunotherapy while, for example, also exhibiting improved performance for imaging or detection. Also provided are therapeutic methods using the conjugates and compositions for treatment of diseases and conditions, including tumors or cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/753,153, filed on Feb. 15, 2018, which is a U.S. National PhaseApplication of International Application No. PCT/US2016/047640, filedAug. 18, 2016, which claims priority from U.S. provisional applicationNo. 62/206,776, filed Aug. 18, 2015, entitled “Combination Therapy withPhotoimmunotherapy and Related Methods,” and from U.S. provisionalapplication No. 62/249,085, filed Oct. 30, 2015, entitled “Compositionsand Methods for Photoimmuotherapy,” the contents of each of which areincorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled751702000403seqlist.txt, created Sep. 16, 2021, which is 9,859 bytes insize. The information in electronic format of the Sequence Listing isincorporated by reference in its entirety.

FIELD

The present disclosure relates to conjugates, compositions and methodsfor use in photoimmunotherapy, such as photoimmunotherapy induced byactivation of a phthalocyanine dye conjugated to a targeting moleculethat binds a protein on cell, for example, an IR700-antibody conjugate.The present disclosure also relates to combination therapies for use incombination with photoimmunotherapy, such as photoimmunotherapy inducedby activation of a phthalocyanine dye conjugated to a targeting moleculethat targets a tumor cell, for example, an IR700-antibody conjugate. Insome embodiments, the phthalocyanine-dye conjugate can be activated byirradiation with near-infrared light. Features of the conjugates,compositions, combinations and methods, including the dose of theconjugate, provide various advantages, such as lower toxicity and/orimproved efficacy. In some embodiments, the disclosure also relates to adual label phthalocyanine-dye conjugate in which the targeting moleculeis conjugated to an additional fluorescent dye, which can be used forphotoimmunotherapy while, for example, also exhibiting improvedperformance for imaging or detection. The disclosure also providestherapeutic methods using the conjugates, compositions and combinationsfor treatment of diseases and conditions, including tumors or cancers.

BACKGROUND

Various therapies are available for treating disease, such as cancer.For example, photoimmunotherapy (PIT) is a method that uses aphotosensitizer conjugated to an antibody or other targeting molecule totarget a cell surface protein in order to permit the targeted killing ofspecific cells. In some cases, PIT can selectively target disease cells,such as tumor cells, and thereby selectively kill such cells withoutdamaging healthy cells. Improved strategies are needed to improvephotoimmunotherapy methods, for example, to increase the efficacy oftreatment. Provided are compositions and methods that meet such needs.

SUMMARY

Provided in some embodiments is a method of treating a disease orcondition in a subject containing using photoimmunotherapy (PIT). Insome embodiments, the method includes administering to the subjecthaving a disease or condition a conjugate containing a phthalocyaninedye linked to a targeting molecule, such as an antibody or anantigen-binding fragment thereof, that binds to a protein on the surfaceof a cell present in the microenvironment of a lesion associated withthe disease or condition. In some embodiments, the conjugate isadministered to effect a systemic exposure that is no more than 75% ofthe total therapeutically effective systemic exposure of the antibody orantigen-binding fragment that is not so conjugated for treating the samedisease or condition as described at the label approved forcommercialization by the regulatory agencies (e.g. FDA, EMA, PDMA). Insome embodiments, after administering the conjugate, the lesion isirradiated at a wavelength of 500 to 900 nm at a dose of at least 1 Jcm⁻² or 1 J/cm of fiber length thereby treating the tumor in thesubject. In some embodiments, the wavelength for irradiation is 600 nmto 850 nm, such as 660 nm to 740 nm.

In some embodiments, the conjugate is administered in a dosing schedulein which: the administration of the conjugate is performed only one timeas a single injection or infusion; or the dosing schedule does notinclude a subsequent dose of the conjugate; or the dosing schedule doesnot include a subsequent dose of the targeting molecule, e.g., amacromolecule, that is not so conjugated.

In some embodiments, the conjugate is administered systemically. In someembodiments, the conjugate is administered intravenously.

In some embodiments, the conjugate is administered to effect a systemicexposure (AUC) that is no more than 60%, no more than 50%, no more than40% or no more than 30% of the therapeutically effective systemicexposure of the antibody or antigen-binding fragment that is not soconjugated for treating the same disease or condition.

In some embodiments, the disease or condition is a tumor, whereby theantibody or an antigen-binding fragment binds to a molecule on thesurface of a cell present in the tumor microenvironment and the tumor isirradiated.

Provided in some embodiments is a method of treating a disease orcondition in a subject using photoimmunotherapy (PIT) wherein thesystemic exposure as measured by the average area under the plasmaconjugate concentration-time curve from time 0 to infinity (AUC[0-inf]or AUC_(0-∞)) for a patient population, such as a sample patientpopulation after administration of the conjugate is between or betweenabout 250 μg/mL*h and 100,000 μg/mL*h, between or between about 500μg/mL*h and 50,000 μg/mL*h, between or between about 500 μg/mL*h and18,000 μg/mL*h, or between or between about 500 μg/mL*h and 10,000μg/mL*h. In some embodiments, the systemic exposure as measured by theaverage area under the plasma conjugate concentration-time curve fromtime 0 to infinity (AUC[0-inf] or AUC_(0-∞)) for a patient population,such as a sample patient population after administration of theconjugate is no more than 100,000 μg/mL*h, no more than 75,000 μg/mL*h,no more than 50,000 μg/mL*h, no more than 40,000 μg/mL*h, no more than30,000 μg/mL*h, no more than 20,000 μg/mL*h, no more than 10,000μg/mL*h, no more than 5,000 μg/mL*h, or no more than 2,500 μg/mL*h.

In some embodiments, the systemic exposure as measured by the averagearea under the plasma conjugate concentration-time curve from time 0 to24 hours (AUC[0-24] or AUC₀₋₂₄) for a patient population, such as asample patient population after administration of the conjugate isbetween or between about 100 μg/mL*h and 25,000 μg/mL*h, between orbetween about 200 μs/mL*h and 10,000 μg/mL*h, between or between about500 μg/mL*h and 5,000 μg/mL*h; or the systemic exposure as measured bythe average area under the plasma conjugate concentration-time curvefrom time 0 to 24 hours (AUC[0-24] or AUC₀₋₂₄) for a patient population,such as a sample patient population after administration of theconjugate is no more than 25,000 μg/mL*h, no more than 15,000 μg/mL*h,no more than 10,000 μg/mL*h, no more than 5,000 μg/mL*h, no more than2,500 μg/mL*h, no more than 1,000 μg/mL*h, or no more than 500 μg/mL*h.

In some embodiments, the conjugate is administered in a dosage rangethat is at least about 10 mg/m² (body surface area of the subject), atleast about 50 mg/m² or at least about 75 mg/m² and is no more than 5000mg/m², no more than 2000 mg/m², no more than 1000 mg/m². In someembodiments, the conjugate is administered in a dosage range that is nomore than 500 mg/m², no more than 250 mg/m², or no more than 200 mg/m².In some embodiments, the conjugate is administered at a dosage that isbetween or between about 100 mg/m² and 1500 mg/m² or 150 mg/m² and 750mg/m². In some embodiments, the conjugate is administered at a dosagethat is or is about 160 mg/m², 320 mg/m², 640 mg/m² or 1280 mg/m².

In some embodiments, the targeting molecule is an antibody or anantigen-binding antibody fragment. In some embodiments, the antibody isan antigen-binding antibody fragment that is a Fab, a single V_(H)domain, a single chain variable fragment (scFv), a multivalent scFv, abispecific scFv or an scFv-CH3 dimer.

In some embodiments, the lesion is irradiated at a wavelength of 690±50nm or at a wavelength of or about 690±20 nm. In some embodiments, thelesion is irradiated at a dose of from or from about 2 J cm⁻² to about400 J cm⁻² or from or from about 2 J/cm fiber length to about 500 J/cmfiber length. In some embodiments, the lesion is irradiated at a dose ofat least or at least about 2 J cm⁻², 5 J cm⁻², 10 J cm⁻², 25 J cm⁻², 50J cm⁻², 75 J cm⁻², 100 J cm⁻², 150 J cm⁻², 200 J cm⁻², 300 J cm⁻², 400 Jcm⁻², or 500 J cm⁻²; or the lesion is irradiated at a dose of at leastor at least about 2 J/cm fiber length, 5 J/cm fiber length, 10 J/cmfiber length, 25 J/cm fiber length, 50 J/cm fiber length, 75 J/cm fiberlength, 100 J/cm fiber length, 150 J/cm fiber length, 200 J/cm fiberlength, 250 J/cm fiber length, 300 J/cm fiber length, 400 J/cm fiberlength or 500 J/cm fiber length.

In some embodiments, the irradiation is carried out or effected betweenor between about 30 minutes and 96 hours after administering theconjugate. In some embodiments, the conjugate is administered in adosing schedule in which the administration of the conjugate isperformed only one time as a single injection or infusion. In someembodiments, the conjugate is administered in a dosing schedule in whichthe dosing schedule does not contain a subsequent dose of the conjugate.In some embodiments, the conjugate is administered in a dosing schedulein which the dosing schedule does not contain a subsequent dose of thetargeting molecule that is not so conjugated. In some embodiments, thedosing schedule is repeated.

In some embodiments, the conjugate is administered systemically. In someembodiments, the conjugate is administered intravenously.

In some embodiments, the phthalocyanine dye has a maximum absorptionwavelength from or from about 600 nm to about 850 nm.

In some embodiments, the phthalocyanine dye is linked directly orindirectly to the targeting molecule. In some embodiments, thephthalocyanine dye includes the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ include a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

In some embodiments, the phthalocyanine dye includes the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ includes a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

In some embodiments, the phthalocyanine dye includes IRDye 700DX(IR700).

In some embodiments, the cell surface protein is selected from amongACTHR, endothelial cell Anxa-1, aminopetidase N, anti-IL-6R,alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin,alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1,B2, BAGE1, BAGE2, B-cell receptor BB1, BB2, BB4, calcitonin receptor,cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14,CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90,CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (CarcinoEmbryonicAntigen), CGRP, chemokine receptors, cell-surface annexin-1,cell-surface plectin-1, Cripto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2glycoprotein, EGFR, EGFRvIII, EMR1, Endosialin, EP2, EP4, EpCAM, EphA2,ET receptors, Fibronectin, Fibronectin ED-B, FGFR, frizzled receptors,GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1 receptor, G-proteincoupled receptors of the Family A (Rhodopsin-like), G-protein coupledreceptors of the Family B (Secretin receptor-like) like), G-proteincoupled receptors of the Family C (Metabotropic GlutamateReceptor-like), GD2, GP100, GP120, Glypican-3, hemagglutinin, Heparinsulfates, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7 antigens,hTERT, IL11-R, IL-13R, ITGAM, Kalikrien-9, Lewis Y, LH receptor, LHRH-R,LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE 4, MART 1, MC1R, Mesothelin,MUC1, MUC16, Neu (cell-surface Nucleolin), Neprilysin, Neuropilin-1,Neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutantp53, p97 melanoma antigen, NTR2, NTR3, p32 (p32/gC1q-R/HABP1), p75,PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptors, PDT, Protease-cleavedcollagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA,PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2,RAMP3 patched, RET receptor, plexins, smoothened, sst1, sst2A, sst2B,sst3, sst4, sst5, substance P, TEMs, T-cell CD3 Receptor, TAG72, TGFBR1,TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM,TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3),TSH receptor, VEGF receptors (VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, andVEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor1, Y1, Y2, Y4, and Y5.

In some embodiments, the cell surface protein is selected from amongHER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2Ra receptor), CD33, CD52, CD133,CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125(CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin,PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelialgrowth factor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCRcomplex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen,IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, Ephrin receptor,Ephrin ligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor, and SK-1antigen.

In some embodiments, the cell surface protein is selected from amongCD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4,LAG3 (CD223), (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184),CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA (PD1 homologue), B7-H4(VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155,CD226, TIGIT, CD96, Galectin 3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM(TNFRSF14), B7-H3 (CD276), Ox40L (TNFSF4), CD137L (TNFSF9, GITRL),B7RP1, ICOS (CD278), ICOSL, KIR, GALS, NKG2A (CD94), GARP, TL1A,TNFRSF25, TMIGD2, BTNL2, Butyrophilin family, CD48, CD244, Siglecfamily, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A),MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family(Killer-cell immunoglobulin-like receptor, LILR family (Leukocyteimmunoglobulin-like receptors, CD85, ILTs, LIRs), SIRPA (Signalregulatory protein alpha), CD47 (IAP), Neuropilin 1 (NRP-1), a VEGFR,and VEGF.

In some embodiments, the antibody or an antigen-binding fragment isselected from among cetuximab, panitumumab, zalutumumab, nimotuzumab,Tositumomab (Bexxar®), Rituximab (Rituxan, Mabthera), Ibritumomabtiuxetan (Zevalin), Daclizumab (Zenapax), Gemtuzumab (Mylotarg),Alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705,B72.3, Bevacizumab (Avastin®), Basiliximab, nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166,dacetuzumab, lucatumumab, SEA-CD40, CP-870, CP-893, MED16469, MEDI6383,MEDI4736, MOXR0916, AMP-224, PDR001, MSB0010718C, rHIgM12B7,Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab(BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 andMNRP1685A or is an antigen-binding fragment thereof.

In some embodiments, the conjugate is selected from amongcetuximab-IR700, panitumumab-IR700, zalutumumab-IR700,nimotuzumab-IR700, Tositumomab-IR700, Rituximab-IR700, Ibritumomabtiuxetan-IR700, Daclizumab-IR700, Gemtuzumab-IR700, Alemtuzumab-IR700,CEA-scan Fab fragment-IR700, OC125-IR700, ab75705-IR700, B72.3-IR700,Bevacizumab-IR700, Basiliximab-IR700, nivolumab-IR700,pembrolizumab-IR700, pidilizumab-IR700, MK-3475-IR700, BMS-936559-IR700,MPDL3280A-IR700, ipilimumab-IR700, tremelimumab-IR700, IMP321-IR700,BMS-986016-IR700, LAG525-IR700, urelumab-IR700, PF-05082566-IR700,TRX518-IR700, MK-4166-IR700, dacetuzumab-IR700, lucatumumab-IR700,SEA-CD40-IR700, CP-870-IR700, CP-893-IR700, MED16469-IR700,MEDI6383-IR700, MEDI4736-IR700, MOXR0916-IR700, AMP-224-IR700,PDR001-IR700, MSB0010718C-IR700, rHIgM12B7-IR700, Ulocuplumab-IR700,BKT140-IR700, Varlilumab-IR700, ARGX-110-IR700, MGA271-IR700,lirilumab-IR700, IPH2201-IR700, AGX-115-IR700, Emactuzumab-IR700,CC-90002-IR700 and MNRP1685A-IR700.

In some embodiments, the targeting molecule is an antibody that iscetuximab or is an antigen-binding fragment thereof or the conjugate iscetuximab-IR700.

In some embodiments, the average area under the plasma conjugateconcentration-time curve from time 0 to infinity (AUC[0-inf] orAUC_(0-∞)) for a patient population, such as a sample patient populationafter administration of the conjugate is between or between about 500μg/mL*h and 18,000 μg/mL*h, between or between about 500 μg/mL*h and10,000 μg/mL*h, between or between about 500 μg/mL*h and 5,000 μg/mL*h,or between or between about 500 μg/mL*h and 2,500 μg/mL*h. In someembodiments, the average area under the plasma conjugateconcentration-time curve from time 0 to 24 hours (AUC[0-24] or AUC₀₋₂₄)for a patient population, such as a sample patient population afteradministration of the conjugate is between or between about 500 μg/mL*hand 8,000 μg/mL*h, between or between about 500 μg/mL*h and 5,000μg/mL*h, between or between about 500 μg/mL*h and 2,000 μg/mL*h orbetween or between about 1000 μg/mL*h and 4,000 μg/mL*h.

In some embodiments, the conjugate is administered in a dosage rangethat is between or between about 75 mg/m² (body surface area of thesubject) and 1500 mg/m², between or between about 75 mg/m² and 1000mg/m², between or between about 75 mg/m² and 500 mg/m² or between orbetween about 75 mg/m² and 225 mg/m². In some embodiments, the conjugateis administered in a dosage range that is at least about or is about 160mg/m², 320 mg/m², 640 mg/m² or 1280 mg/m².

Provided in some embodiments is a method of treating a disease lesion ina subject, that includes: a) intravenously administering to a subjecthaving a lesion associated with a disease or condition a cetuximab-IR700conjugate, wherein the conjugate is administered in an amount that is oris about 640 mg/m²; and b) after administering the conjugate,irradiating the lesion at a wavelength of 690±20 nm at a dose of atleast or about at least or about 50 J cm⁻² or 100 J/cm of fiber length,thereby treating the disease or condition in the subject.

In some embodiments, the conjugate is administered in a dosing schedulein which: the administration of the conjugate is performed only one timeas a single injection or infusion; or the dosing schedule does notinclude a subsequent dose of the conjugate; or the dosing schedule doesnot include a subsequent dose of the targeting molecule that is not soconjugated.

In some embodiments, the irradiation is carried out 24 hours±3 hours,such as 24 hours±2 hours, after administering the conjugate.

In some embodiments, the lesion is a tumor and the disease or conditionis a tumor or a cancer.

In some embodiments, the lesion is a tumor that is a superficial tumor.In some embodiments, the tumor is less than 10 mm thick. In someembodiments, irradiation is carried out using a microlens-tipped fiberfor surface illumination. In some embodiments, the light irradiationdose is from or from about 5 J/cm² to about 200 J/cm².

Provided in some embodiments is a method for treating a superficialtumor with photoimmunotherapy, that includes illuminating an superficialtumor in a subject with a microlens-tipped fiber for surfaceillumination with a light dose of from or from about 5 J/cm² to about200 J/cm², wherein the tumor is associated with a phototoxic agent thatincludes a targeting molecule bound to a cell surface molecule of thetumor. In some embodiments, the light irradiation dose is or is about 50J/cm².

In some embodiments, the lesion is a tumor that is an interstitialtumor. In some embodiments, the tumor is greater than 10 mm deep or is asubcutaneous tumor. In some embodiments, irradiation is carried outusing cylindrical diffusing fibers that includes a diffuser length of0.5 cm to 10 cm and spaced 1.8±0.2 cm apart. In some embodiments, thelight irradiation dose is from or from about 20 J/cm fiber length toabout 500 J/cm fiber length.

Provided in some embodiments is a method for treating an interstitialtumor with photoimmunotherapy, that includes illuminating aninterstitial tumor in a subject with cylindrical diffusing fibers thatincludes a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cmapart with a light dose of or about 100 J/cm fiber length or with afluence rate of or about 400 mW/cm, wherein the tumor is associated witha phototoxic agent that includes a targeting molecule bound to a cellsurface molecule of the tumor.

In some embodiments, the light irradiation dose is from or from about 50J/cm fiber length to about 300 J/cm fiber length. In some embodiments,the light irradiation dose is or is about 100 J/cm fiber length.

In some embodiments, the tumor is greater than 10 mm deep or is asubcutaneous tumor. In some embodiments, the cylindrical diffusingfibers are placed in a catheter positioned in the tumor 1.8±0.2 cmapart. In some embodiments, the catheter is optically transparent.

In some embodiments, greater than 6 hours prior to illuminating thetumor, the subject has been administered the phototoxic agent thatincludes the targeting molecule, wherein the phototoxic agent associateswith the tumor. In some embodiments, the phototoxic agent has beenpreviously administered to the subject greater than or greater thanabout 12 hours, 24 hours, 26 hours, 48 hours, 72 hours or 96 hours priorto illuminating the tumor. In some embodiments, the phototoxic agent isa phthalocyanine dye-targeting molecule conjugate. In some embodiments,the phthalocyanine dye is IR700.

In some embodiments, in any of the methods for treating provided herein,the dosing schedule is repeated, whereby steps (a) and (b) are repeated.In some embodiments, the dosing schedule is repeated if residual lesionremains after a prior treatment with the conjugate. In some embodiments,the method additionally includes assessing the subject for the presenceof a residual lesion and if residual lesion remains repeating the dosingschedule. In some embodiments, the dosing schedule is repeated if aresidual lesion remains at a time that is more than or about or 1 week,2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after initiationof the prior administration of the conjugate. In some embodiments, thedosing schedule is repeated if a residual lesion remains at or about 4weeks after initiation of the prior administration of the conjugate.

In some embodiments, the conjugate contains 1 to 100, 1 to 10 or 2 to 5phthalocyanine dye molecules per targeting molecule.

In some embodiments, the method does not contain administration of anadditional therapeutic agent or anti-cancer treatment. In someembodiments, the method contains administration of an additionaltherapeutic agent or anti-cancer treatment. In some embodiments, theanti-cancer treatment contains radiation therapy.

In some embodiments, the additional therapeutic agent is an anti-canceragent or an immune modulating agent. In some embodiments, the additionaltherapeutic agent is an immune modulating agent is an immune checkpointinhibitor. In some embodiments, the immune checkpoint inhibitorspecifically binds a molecule selected from among CD25, PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA. In some embodiments,the immune checkpoint inhibitor is and antibody or antigen-bindingfragment, a small molecule or a polypeptide. In some embodiments, theimmune checkpoint inhibitor is selected from among nivolumab,pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab,tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab,lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916,AMP-224, and MSB001078C, or is an antigen-binding fragment thereof.

In some embodiments, the immune modulating agent is administered priorto irradiating the lesion or tumor. In some embodiments, the immunemodulating agent is administered greater than or greater than about 30minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 96hours, one week, two weeks, three weeks or one month prior toirradiating the tumor.

In some embodiments, the provided methods include continuedadministration of the immune modulating agent subsequent to theirradiation three times a week, two times a week, once every week, onceevery two weeks, once every three weeks or once a month.

Provided in some embodiments is a method of treating a tumor in asubject that includes: a) administering to a subject an immunemodulating agent; b) administering to the subject a therapeuticallyeffective amount of a conjugate that includes a phthalocyanine dyelinked to a targeting molecule capable of binding to a molecule on thesurface of a cell present in the microenvironment of a tumor; and c)greater than 12 hours after administering the immune modulating agent,irradiating the tumor at a wavelength that renders the conjugatecytotoxic, thereby treating the tumor. In some embodiments, the immunemodulating agent is administered greater than or greater than about 24hours, 48 hours, 96 hours, one week, two weeks, three weeks or one monthprior to irradiating the tumor.

In some embodiments, the conjugate binds to a protein on the surface ofa cell present in the microenvironment of the tumor. In some embodimentsof the provided methods, step c) of irradiating the tumor is carried outeither i) after administration of the immune modulating agent and afteradministration of the conjugate or ii) only after administration of theconjugate.

In some embodiments, the conjugate is administered prior to,simultaneously or subsequently to administration of theimmune-modulating agent. In some embodiments, the conjugate isadministered after administering the immune modulating agent but priorto irradiating the tumor. In some embodiments, the conjugate isadministered from or from about 12 hours to 48 hours prior toirradiating the tumor and the immune modulating agent is administeredfrom or from about 12 hours to about 1 month prior to irradiating thetumor.

In some embodiments, the immune modulating agent is an immune checkpointinhibitor. In some embodiments, the immune checkpoint inhibitorspecifically binds a molecule selected from among CD25, PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA. In some embodiments,the immune checkpoint inhibitor is and antibody or antigen-bindingfragment, a small molecule or a polypeptide. In some embodiments, theimmune checkpoint inhibitor is selected from among nivolumab,pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab,tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab,lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916,AMP-224, and MSB001078C, or is an antigen-binding fragment thereof ofany of the foregoing.

In some embodiments, the immune modulating agent that is a demethylatingagent that upregulates expression of a tumor associated antigen (TAA) oris a cytokine.

In some embodiments, the provided methods include continuedadministration of the immune modulating agent subsequent to theirradiation three times a week, two times a week, once every week, onceevery two weeks, once every three weeks or once a month.

Provided in some embodiments is a method of treating a tumor in asubject that includes: a) administering to a subject an immunemodulating agent that enhances the expression of a molecule on thesurface of a cell present in the microenvironment of the tumor; b)administering to the subject a therapeutically effective amount of aconjugate that includes a phthalocyanine dye linked to a targetingmolecule that is capable of binding to the cell surface molecule; and c)greater than 5 minutes after administering the conjugate, irradiatingthe tumor at a wavelength that renders the conjugate cytotoxic, therebytreating the tumor.

In some embodiments, the immune modulating agent is a cytokine or is anagent that induces increased expression of a cytokine in the tumormicroenvironment. In some embodiments, the cytokine is interferon gamma.

In some embodiments, the molecule on the surface of the cells isselected from CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB,GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28and VISTA. In some embodiments, the molecule on the surface of the cellis PD-L1.

In some embodiments, the targeting molecule is an immune checkpointinhibitor.

In some embodiments, the targeting molecule is an antibody or antibodyfragment, a small molecule or a polypeptide. In some embodiments, thetargeting molecule is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing.

Provided in some embodiments is a method of treating a tumor in asubject that includes: a) administering to a subject a conjugate thatincludes a phthalocyanine dye linked to a targeting molecule capable ofbinding a cell surface molecule on a cell in the microenvironment of thetumor; b) greater than 5 minutes after administering the conjugate,irradiating the tumor at a wavelength that renders the conjugatecytotoxic, wherein the treatment of the tumor with the conjugatefollowed by light irradiation increases the presence ofimmunosuppressive cells in the tumor or increases the expression ofimmunosuppressive markers at the tumor; and c) administering to thesubject a therapeutically effective amount of an immune modulating agentcapable of reducing the amount or activity of immunosuppressive cells inthe tumor or capable of blocking the activity of the immunosuppressivemarker or reducing the activity of a tumor promoting cell in the tumoror capable of blocking the activity of the tumor promoting marker.

In some embodiments, the phthalocyanine dye is a first dye and theimmune modulating agent includes a conjugate that includes a secondphthalocyanine dye conjugated to an immune modulating agent capable ofbinding to the immunosuppressive cell or a tumor promoting cell. In someembodiments, the first and second phthalocyanine dye is the same ordifferent.

In some embodiments, the immune modulating agent is an immune checkpointinhibitor. In some embodiments, the immune modulating agent specificallybinds a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4,LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3,B7-H4, BTLA, HVEM, CD28 and VISTA.

In some embodiments, the immune modulating agent is an antibody orantibody fragment, a small molecule or a polypeptide.

In some embodiments, the immune modulating agent is not an anti-CTLA4antibody.

In some embodiments, the immune modulating agent is selected from amongnivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A,ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518,dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MED14736,MOXR0916, AMP-224, and MSB001078C, or is an antigen-binding fragmentthereof of any of the foregoing.

Provided in some embodiments is a method of treating a tumor in asubject that includes: a) administering to a subject a conjugate thatincludes a phthalocyanine dye linked to a targeting molecule capable ofbinding to a molecule on the surface of a cell present in themicroenvironment of the tumor; b) greater than 5 minutes afteradministering the conjugate, irradiating the tumor at a wavelength thatrenders the conjugate cytotoxic, wherein the treatment of the tumor withthe conjugate followed by light irradiation primes activation of immunecells; and c) administering to the subject a therapeutically effectiveamount of an immune modulating agent capable of increasing the activityof the immune cell.

In some embodiments, the immune cell is an antigen presenting cell. Insome embodiments, the immune cell is a dendritic cell. In someembodiments, the immune modulating agent is selected from among GM-CSF,CpG-ODN (CpG oligodeoxynucleotides), lipopolysaccharide (LPS),monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein,imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys,complete freund's adjuvant (CFA), alpha-galactosylceramide, RC-529,MDF2β, Loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03,Flagellin, Resiquimod, DAP (diaminopimelic acid), MDP (muramyldipeptide) and CAF01 (cationic adjuvant formulation-01). In someembodiments, the immune modulating agent is a Toll-like receptor (TLR)agonist, an adjuvant or a cytokine. In some embodiments, the immunemodulating agent is a TLR agonist and the TLR agonist is TLR agonist isa TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. Insome embodiments, the TLR agonist is selected from among triacylatedlipoprotein, diacylated lipopeptide, lipoteichoic acid, peptidoglycan,zymosan, Pam3CSK4, dsRNA, polyI:C, Poly G10, Poly G3, CpG, 3M003,flagellin, lipopolysaccharide (LPS) Leishmania homolog of eukaryoticribosomal elongation and initiation factor 4a (LeIF), MEDI9197, SD-101,and imidazoquinoline TLR agonists.

In some embodiments, the immune modulating agent is a cytokine and thecytokine is IL-4, TNF-α, GM-CSF or IL-2. In some embodiments, the immunemodulating agent is administered prior to, simultaneously with or afterthe irradiation. In some embodiments, the immune modulating agent isadministered no more than 5 minutes, 30 minutes, 60 minutes, 2 hours, 6hours, 12 hours or 24 hours after the irradiation. In some embodiments,the immune modulating agent is administered no more than 5 minutes, 30minutes, 60 minutes, 2 hours, 6 hours, 12 hours or 24 hours before theirradiation.

In some embodiments, the targeting molecule binds to molecule or proteindirectly or indirectly. In some embodiments, the targeting molecule is asecond binding molecule that binds to a first binding molecule, saidfirst binding molecule being capable of binding to the molecule orprotein. In some embodiments, the targeting molecule is a secondaryantibody.

In some embodiments, the phthalocyanine dye has a maximum absorptionwavelength from or from about 600 nm to about 850 nm.

In some embodiments, the phthalocyanine dye is covalently ornon-covalently linked to the targeting molecule. In some embodiments,the phthalocyanine dye includes a linker that includes a reactive groupfor attachment of the dye to the targeting molecule.

In some embodiments, the phthalocyanine dye includes the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ includes a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

In some embodiments, the phthalocyanine dye includes the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ includes a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

In some embodiments, the phthalocyanine dye includes IRDye 700DX(IR700).

In some embodiments, the conjugate is administered at a dose from orfrom about 50 mg/m² to about 5000 mg/m², from about 250 mg/m² to about2500 mg/m², from about 750 mg/m² to about 1250 mg/m² or from about 100mg/m² to about 1000 mg/m².

In some embodiments, the tumor is a cancer. In some embodiments, thecancer is a cancer located at the head and neck, breast, liver, colon,ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder,stomach, esophagus, peritoneum, or lung. In some embodiments, the tumoris a sarcoma or carcinoma. In some embodiments, the tumor is a carcinomathat is a squamous cell carcinoma, basal cell carcinoma oradenocarcinoma. In some embodiments, the tumor is a carcinoma that is acarcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach,prostate, cervix, esophagus or head and neck.

In some embodiments, the tumor is irradiated at a wavelength of 600 nmto 850 nm at a dose of at least 1 J cm⁻² or at least 1 J/cm fiberlength. In some embodiments, the tumor is irradiated at a wavelength of690 nm±50 nm or at a wavelength of or about 690±20 nm.

In some embodiments, the method reduces the size or volume of the tumorby at least 30%, at least 40%, at least 50%, at least 60%, at least 70%,at least 80% at least 90% or more within one month of the irradiationcompared to the size or volume of the tumor prior to the administrationand irradiation.

In some embodiments, the method of PIT treatment using thephthalocyanine dye conjugate, such as in accord with any of the methodsabove or provided herein, results in an improvement of a disorder- orcancer-related parameter in a population of treated subjects compared toa similarly situated population of subjects treated with the antibody orantigen-binding antibody fragment that is not conjugated. In someembodiments, the parameter is selected from one or more of: a) objectiveresponse rate (ORR); b) progression free survival (PFS); c) overallsurvival (OS); d) reduction in toxicity; e) tumor response; or f)quality of life.

In some embodiments, the parameter is improved by at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100% or more.

In some embodiments, the method of PIT treatment using thephthalocyanine dye conjugate, such as in accord with any of the methodsabove or provided herein, results in an objective response rate (ORR)that is at least 15%, at least 25%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or more in a population of treatedsubjects.

In some embodiments, the conjugate contains a phthalocyanine dye as afirst dye and further contains a second fluorescent dye linked to thetargeting molecule that is different than the first dye. In someembodiments, the second fluorescent dye exhibits one or more spectralproperties selected from among fluorescent quantum yield in water,extinction coefficient, Stokes shift, absorption and emission at longwavelength and photostability that is greater compared to thecorresponding spectral property of the first dye. In some embodiments,the lesion or tumor emits a fluorescence signal from the secondfluorescent dye to effect detection of the presence of the conjugate atthe lesion or tumor in the subject. In some embodiments, the providedmethod further includes imaging the lesion or tumor in the subject byirradiating or illuminating the tumor at a wavelength capable of beingabsorbed by the second dye.

In some embodiments, the first dye is IR700. In some embodiments, thesecond dye is not IR700. In some embodiments, the second dye is selectedfrom among hydroxycoumarin, Cascade Blue, Dylight 405, Pacific Orange,Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488, BODIPY 493,2.7-Diochlorofluorescien, ATTO 488, Chromeo 488, Dylight 488, HiLyte488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3,TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564,Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610,Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633,CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, AlexaFluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7,Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565,Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

In some embodiments, the first dye is IR700 and the conjugate contains 1to 10 or 1 to 5 second dye molecules per targeting molecule.

In some embodiments, the second dye exhibits a Stokes shift that isgreater than 15 nm, greater than 20 nm, greater than 30 nm, greater than40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm,greater than 80 nm, greater than 90 nm or greater than 100 nm.

In some embodiments, the second dye has a quantum yield in water that isgreater than 10%, greater than 15%, greater than 20% or greater than25%, greater than 30%, greater than 40%, greater than 50% or greater.

In some embodiments, the second dye has an absorption and emissionwavelength in the spectrum between or between about 650 nm and 950 nm,between or between about 700 nm and 1000 nm, or between or between about1000 nm and 1700 nm.

In some embodiments, the first dye and second dye do not exhibit anoverlapping emission and absorption spectra. In some embodiments, thesecond dye is selected from among ICG, IRDye 680, Alexa Fluor 750,Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye800. In some embodiments, the second dye is Alexa Fluor 488, IRDye 680,IRDye 800 or Dylight 755.

In some embodiments, the method further includes irradiating orilluminating the tumor at a wavelength capable of being absorbed by thesecond dye, thereby imaging the subject. In some embodiments, theirradiation or illumination of the tumor is performed with a deviceselected from among a hand-held ultraviolet lamp, a mercury lamp, axenon lamp, a laser, a laser diode or an imaging device. In someembodiments, the LED imaging device contains a near-infrared (NIR)diode.

Provided in some embodiments is a composition containing a conjugatecontaining a phthalocyanine dye linked to an antibody or antigen-bindingantibody fragment that binds to a molecule on the surface of a cellpresent in the microenvironment of a lesion, such as the tumormicroenvironment. In some embodiments, the composition is formulated forsingle dosage administration of the conjugate in an amount that isbetween or between about 100 mg and 1200 mg. In some embodiments, thecomposition is formulated for single dosage administration of an amountbetween or between about 100 mg and 500 mg or between or between about200 mg and 400 mg. In some embodiments, the composition is formulatedfor single dosage administration of an amount between or between about500 mg and 1500 mg, 800 mg and 1200 mg or 1000 mg and 1500 mg.

In some embodiments, the volume of the composition is between or betweenabout 10 mL and 500 mL or 50 mL and 250 mL. In some embodiments, thevolume of the composition is at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL,75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL or 500 mL. In someembodiments, the volume of the composition is between or between about10 mL and 1000 mL or 50 mL and 500 mL; or the volume of the compositionis at least 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL,200 mL, 250 mL, 300 mL, 400 mL, 500 mL or 1000 mL.

Provided in some embodiments is an article of manufacture, including aplurality of sealable containers, each individually containing afraction of a single administration dose of a composition containing aconjugate containing a phthalocyanine dye linked to an antibody orantigen-binding antibody fragment that binds to a molecule on thesurface of a cell present in the microenvironment of a lesion, such asthe tumor microenvironment. In some embodiments, the combined amount ofthe conjugate in the plurality of sealable containers is between orbetween about 100 mg and 1500 mg, or 100 mg and 1200 mg. In someembodiments, the article of manufacture contains packaging material anda label or package insert containing instructions for combining thecontents of the plurality of vials to prepare a single dosageformulation of the composition.

In some embodiments, the combined amount of the conjugate in theplurality of sealable containers is between or between about 100 mg and1200 mg. In some embodiments, the combined amount of the conjugate inthe plurality of sealable container is between or between about 100 mgand 500 mg, between or between about 200 mg and 400 mg, between orbetween about 500 mg and 1500 mg, between or between about 800 mg and1200 mg or between or between about 1000 mg and 1500 mg.

In some embodiments, the lesion is a tumor.

Provided in some embodiments is a conjugate, that includes aphthalocyanine dye linked to an antibody or antigen-binding fragmentthat is an immune modulating agent. In some embodiments, the immunemodulating agent is an immune checkpoint inhibitor.

In some embodiments, the immune modulating agent is an antibody orantigen binding fragment that binds to the surface of a tumor, tumorcell or cancer cell. In some embodiments, the immune modulating agentspecifically binds a molecule selected from among CD25, PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA. In some embodiments theimmune modulating agent is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing. Insome embodiments, the immune modulating agent is an antibody or antibodyfragment that binds to PD-L1.

In some embodiments, the immune modulating agent is an antibody selectedfrom BMS-935559, MEDI4736, MPDL3280A and MSB0010718C, or anantigen-binding fragment thereof.

Provided in some embodiments is a conjugate that contains a targetingmolecule linked to at least a first and second fluorescent dye. In someembodiments, the first fluorescent dye is a phthalocyanine dye capableof exhibiting phototoxicity.

In some embodiments, the conjugate has the formula:

[D₁-(L₁)_(n)]_(p)-A-[(L₂)_(m)-D₂]_(o), wherein:

A is a targeting molecule that can bind to a molecule on the surface ofa cell;

L₁ and L₂ are each an independently selected linker, which can be thesame or different;

n and m are independently 1 or 2;

D₁ is a first dye that is the phthalocyanine dye capable of exhibitingphototoxicity;

D₂ is a second dye that is a fluorescent dye, wherein D₂ is differentthan D₁;

p is 1 to 10; and

o is 1 to 10.

In some embodiments, the targeting molecule is an antibody or anantigen-binding antibody fragment.

In some embodiments, the cell surface molecule contains an antigen, apolypeptide, a lipid, or a carbohydrate or a combination of thesemolecules.

In some embodiments, the cell surface molecule is selected from amongACTHR, endothelial cell Anxa-1, aminopetidase N, anti-IL-6R,alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin,alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1,B2, BAGE1, BAGE2, B-cell receptor BB1, BB2, BB4, calcitonin receptor,cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14,CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90,CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (CarcinoEmbryonicAntigen), CGRP, chemokine receptors, cell-surface annexin-1,cell-surface plectin-1, Cripto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2glycoprotein, EGFR, EGFRvIII, EMR1, Endosialin, EP2, EP4, EpCAM, EphA2,ET receptors, Fibronectin, Fibronectin ED-B, FGFR, frizzled receptors,GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1 receptor, G-proteincoupled receptors of the Family A (Rhodopsin-like), G-protein coupledreceptors of the Family B (Secretin receptor-like) like), G-proteincoupled receptors of the Family C (Metabotropic GlutamateReceptor-like), GD2, GP100, GP120, Glypican-3, hemagglutinin, Heparinsulfates, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7 antigens,hTERT, IL11-R, IL-13R, ITGAM, Kalikrien-9, Lewis Y, LH receptor, LHRH-R,LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE 4, MART 1, MC1R, Mesothelin,MUC1, MUC16, Neu (cell-surface Nucleolin), Neprilysin, Neuropilin-1,Neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutantp53, p97 melanoma antigen, NTR2, NTR3, p32 (p32/gC1q-R/HABP1), p75,PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptors, PDT, Protease-cleavedcollagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA,PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2,RAMP3 patched, RET receptor, plexins, smoothened, sst1, sst2A, sst2B,sst3, sst4, sst5, substance P, TEMs, T-cell CD3 Receptor, TAG72, TGFBR1,TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM,TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3),TSH receptor, VEGF receptors (VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, andVEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor1, Y1, Y2, Y4, and Y5.

In some embodiments, the cell surface molecule is selected from amongHER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2Ra receptor), CD33, CD52, CD133,CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125(CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin,PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelialgrowth factor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCRcomplex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen,IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, Ephrin receptor,Ephrin ligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor, and SK-1antigen.

In some embodiments, the cell surface molecule is selected from amongCD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4,LAG3 (CD223), (HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184),CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA (PD1 homologue), B7-H4(VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155,CD226, TIGIT, CD96, Galectin 3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM(TNFRSF14), B7-H3 (CD276), Ox40L (TNFSF4), CD137L (TNFSF9, GITRL),B7RP1, ICOS (CD278), ICOSL, KIR, GALS, NKG2A (CD94), GARP, TL1A,TNFRSF25, TMIGD2, BTNL2, Butyrophilin family, CD48, CD244, Siglecfamily, CD30, CSF1R, MICA (MHC class I polypeptide-related sequence A),MICB (MHC class I polypeptide-related sequence B), NKG2D, KIR family(Killer-cell immunoglobulin-like receptor, LILR family (Leukocyteimmunoglobulin-like receptors, CD85, ILTs, LIRs), SIRPA (Signalregulatory protein alpha), CD47 (IAP), Neuropilin 1 (NRP-1), a VEGFR,and VEGF.

In some embodiments, the targeting molecule is an antibody or anantigen-binding fragment that is selected from among cetuximab,panitumumab, zalutumumab, nimotuzumab, Tositumomab (Bexxar®), Rituximab(Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin), Daclizumab(Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fab fragment,OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab (Avastin®),Basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559,MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525,urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab,SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916,AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab(CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201,AGX-115, Emactuzumab, CC-90002 and MNRP1685A or is an antigen-bindingfragment thereof.

In some embodiments, the targeting molecule is not or does not include ananocarrier. In some embodiments, the targeting molecule is not or doesnot include a virus-like particle, a nanoparticle, a liposome, a quantumdot, or a combination thereof.

In some embodiments, the first dye that is a phthalocyanine dye that hasa maximum absorption wavelength from or from about 600 nm to about 850nm.

In some embodiments, the first dye that is a phthalocyanine dye containsthe formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ contains a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

In some embodiments, the first dye that is a phthalocyanine dye containsthe formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ contains a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

In some embodiments, the first dye that is a phthalocyanine dye containsIRDye 700DX (IR700).

In some embodiments, the second fluorescent dye exhibits one or morespectral properties selected from among fluorescent quantum yield inwater, extinction coefficient, Stokes shift, absorption and emission atlong wavelength and photostability that is greater compared to thecorresponding spectral property of the first dye.

In some embodiments, the second dye is not IR700. In some embodiments,the second dye is selected from among hydroxycoumarin, Cascade Blue,Dylight 405, Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green,Alexa Fluor 488, BODIPY 493, 2.7-Diochlorofluorescien, ATTO 488, Chromeo488, Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X,CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555,Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine,Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, AlexaFluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750,Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot®800.

In some embodiments, the first dye is IR700 and the conjugate contains 1to 10 or 1 to 5 second dye molecules per targeting molecule.

In some embodiments, the second dye exhibits a Stokes shift that isgreater than 15 nm, greater than 20 nm, greater than 30 nm, greater than40 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm,greater than 80 nm, greater than 90 nm or greater than 100 nm.

In some embodiments, the second dye has a quantum yield in water that isgreater than 10%, greater than 15%, greater than 20% or greater than25%, greater than 30%, greater than 40%, greater than 50% or greater.

In some embodiments, the second dye has an absorption and emissionwavelength in the spectrum between or between about 650 nm and 950 nm,between or between about 700 nm and 1000 nm, between or between about1000 nm and 1700 nm.

In some embodiments, the first dye and second dye do not exhibit anoverlapping emission and absorption spectra.

In some embodiments, the second dye is selected from among ICG, IRDye680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790,Dylight 800 and IRDye 800. In some embodiments, the second dye is AlexaFluor 488, IRDye 680, IRDye 800 or Dylight 755.

Provided in some embodiments is a composition, containing any of theconjugates described herein. In some embodiments, the compositionfurther contains a pharmaceutically acceptable excipient.

Provided in some embodiments is a method of treating a disease orcondition in a subject that includes: a) administering to the subject atherapeutically effective amount of any of the conjugates orcompositions described herein, wherein the conjugate binds to a cellpresent in the microenvironment of a lesion associated with a disease orcondition; and b) after administering the conjugate, irradiating thelesion at one or more wavelengths to induce phototoxic activity of theconjugate, thereby treating the disease or condition.

Provided in some embodiments is a method of treating a disease or acondition, such as a tumor in a subject using photoimmunotherapy (PIT),that includes administering to the subject a therapeutically effectiveamount of any of the conjugates or compositions described herein. Insome embodiments, the method includes irradiating the tumor at awavelength of 660 nm to 740 nm at a dose of at least 1 J cm⁻² or 1 J/cmof fiber length, thereby treating the disease or condition in thesubject.

Provided in some embodiments is a method of treating a disease orcondition in a subject that includes: a) administering to the subject atherapeutically effective amount of any of the conjugates orcompositions described herein, wherein the conjugate binds to a cellpresent in the microenvironment of a lesion associated with a disease orcondition; and b) after administering the conjugate, irradiating thelesion at one or more wavelengths to induce phototoxic activity of thefirst dye of the conjugate and a fluorescent signal of the second dye ofthe conjugate.

In some embodiments, the provided methods include irradiating the lesionat a wavelength that is from or from about 400 to about 900 nm at a doseof at least 1 J cm⁻² or 1 J/cm of fiber length. In some embodiments, theprovided methods include irradiating the lesion with a singlewavelength. In some embodiments, the provided methods includeirradiating the lesion at two different wavelengths, simultaneously orsequentially, wherein one wavelength induces the phototoxic activity andthe other wavelength induces the fluorescent signal.

In some embodiments, the disease or condition is a tumor.

In some embodiments, the provided methods include irradiating the tumorat a wavelength of 660 nm to 740 nm and at a dose of at least 1 J cm⁻²,thereby treating the tumor in the subject.

In some embodiments, the tumor is a cancer. In some embodiments, thecancer is a cancer located at the head and neck, breast, liver, colon,ovary, prostate, pancreas, brain, cervix, bone, skin, eye, bladder,stomach, esophagus, peritoneum, or lung. In some embodiments, the tumoris a sarcoma or carcinoma. In some embodiments, the tumor is a carcinomathat is a squamous cell carcinoma, basal cell carcinoma oradenocarcinoma. In some embodiments, the tumor is a carcinoma that is acarcinoma of the bladder, pancreas, colon, ovary, lung, breast, stomach,prostate, cervix, esophagus or head and neck.

In some embodiments, prior to administration of the conjugate, thetargeting molecule is administered to the subject. In some embodiments,the targeting molecule is administered up to 96 hours prior toadministration of the conjugate. In some embodiments, the targetingmolecule is administered at a dose within a range from or from about 10mg/m² to about 500 mg/m². In some embodiments, the targeting molecule isan antibody or antigen binding fragment, such as cetuximab. In someexamples, cetuximab is administered to the subject prior to theadministration of the conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the dose response curves of cetuximab and cetuximab-IRDye700DX conjugate for the inhibition of EGFR phosphorylation in A431cells.

FIG. 2 shows the dose response curve for photoimmunotherapy (PIT) withthe cetuximab-IRDye 700DX conjugate using 8 J/cm² of 690 nm light onA431 cells.

FIG. 3 shows the comparison of dose response curves for thephotoimmunotherapy (PIT) with the cetuximab-IRDye 700DX conjugate using16 J/cm² versus 32 J/cm² of 690 nm light on BxPC3 cells.

FIG. 4 shows the schematic structure for the dual-labeledpanitumumab-IR700DX-Alexa-488 conjugate.

FIGS. 5A and 5B show the progression of cell death afterphotoimmunotherapy (PIT) induced by different conjugates at variouslight dosages (0-16 J/cm²) on BxPC3 pancreatic cancer cells. FIG. 5Ashows cells death after PIT using single-labeled panitumumab-IR700conjugate. FIG. 5B shows cell death after PIT using a dual-labeledpanitumumab-IR700-Alexa-488 conjugate.

FIG. 6A shows the duration of exposure of Cetuximab-IRDye 700DX to 500Lux white fluorescent light or green LED light and its effect on solubleaggregate formation.

FIG. 6B shows the effect of pre-exposure of Cetuximab-IRDye 700DX towhite fluorescent light or green LED light on BxPC3 PIT activity.

FIG. 6C shows the effect of percent Cetuximab-IRDye 700DX solubleaggregate formation on PIT activity.

FIG. 7 shows the PIT killing activity with sequential staining usingCetuximab and donkey anti-human-IRDye 700DX (DxHu IR700) secondaryantibody.

FIG. 8A shows the light-dependent killing of BxPC3 cells withbiotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye700DX (mSA IR700).

FIG. 8B shows the specificity of PIT with biotinylated cetuximabpre-complexed with monomeric streptavidin-IRDye 700DX (mSA IR700).

FIG. 8C shows the effect of monomeric streptavidin-IRDye 700DXpre-exposure to white light on the PIT killing activity withbiotinylated Cetuximab in BxPC3 cells.

FIG. 9A shows the antibody dose-dependent killing of 4T1 cells withdirectly conjugated anti-EpCAM-IRDye 700DX.

FIG. 9B shows the specificity of anti-EpCAM-IRDye 700DX PIT killingactivity.

FIG. 10 shows the Fc receptor-specific killing of THP1 cells byCetuximab-IRDye 700DX.

FIG. 11A shows the specificity of EGF-IRDye 700DX light-dependentkilling in A431 cells.

FIG. 11B shows the effect of EGF-IRDye 700DX pre-exposure to differenttypes of light on light-dependent killing in A431 cells.

FIG. 12A shows the light-dependent killing of BxPC3 cells using CholeraToxin B-IRDye 700DX.

FIG. 12B shows the specificity of Cholera Toxin B-IRDye 700DXlight-activated killing.

FIG. 12C shows the effect of pre-exposure of Cholera Toxin B-IRDye 700DXto different wavelengths of light on light-activated killing in BxPC3cells.

FIG. 13A shows the light-dependent killing of Vero cells with Influenzavirus (X-31)-IRDye 700DX.

FIG. 13B shows the effect of pre-exposure of influenza virus(X-31)-IRDye 700DX to white light vs. green light on photo-activatedcell killing.

FIG. 14A shows the effect of light dose on SNA-IRDye 700DX killingactivity in BxPC3 cells.

FIG. 14B shows the effect of sialidase treatment on the specificity ofSNA-IRDye 700DX binding to cells.

FIG. 15 shows the PIT killing of S. aureus by Cetuximab-IRDye 700DX incombination with laser illumination.

FIG. 16 shows the PIT of influenza virus particles using pre-complexedmouse anti-influenza virus (H3N2) with GtxMs Fab-IRDye 700DX.

FIG. 17 shows the light-dependent killing of influenza virus infectedcells with Mouse anti-influenza virus (H3N2) and Goat anti-Mouse IRDye700DX (GtxMs-IR700).

FIG. 18 shows the PIT killing of rat embryonic dorsal root ganglion(DRG) neurons using Cholera Toxin B-IRDye 700DX.

FIG. 19A shows the effect of IFNgamma treatment on the percent death ofBxPC3 cells.

FIG. 19B shows the effect of IFNgamma treatment on PD-L1 expression inBxPC3 cells.

FIG. 19C shows the effect of IFNgamma treatment on anti-PD-L1 IRDye700DX PIT killing activity in BxPC3 cells.

FIG. 20A shows the effect of PIT treatment in A431 and FaDu cells usingCetuximab-IRDye 700DX on the amount of HMGB1 detected in extracellularsolution.

FIG. 20B shows the upregulation of dendritic cell (DC) maturationmarkers on immature dendric cells (iDCs) co-cultured with tumorssubjected to PIT via cetuximab-IRDye 700DX.

FIG. 20C shows the effect of activating antigen-presenting cells byPIT-treated A431 or FaDu cells (treated using Cetuximab-IRDye 700DX andin the presence of light irradiation) or non-PIT treated A431 or FaDucells (treated using Cetuximab-IRDye 700DX but with no lightirradiation) as assessed by the expression of the exemplary activationmarker CD86.

FIG. 21 shows the effect on activation of dendritic cells by primingdendritic cells with PIT-treated tumor cells (treated usingCetuximab-IRDye 700DX) or non-PIT treated tumor cells (treated usingCetuximab-IRDye 700DX but with no light irradiation) followed by theirstimulation with an immune modulator (Poly I:C) as assessed by theexpression of exemplary activation markers.

FIG. 22A shows the UV-Vis spectrum of cetuximab-IRDye 700DX-Alexa Flour488 (CTX700-ALX488).

FIG. 22B shows the UV-Vis spectrum of CTX-700-IRDye 800CW.

FIG. 22C shows the UV-Vis spectrum of CTX700-Dylight 755.

FIG. 23A shows the PIT killing activity of Cetuximab-IRDye 700DX with orwithout DyLight 755 in BxPC3 cells at various concentrations and lightdoses.

FIG. 23B shows the PIT killing activity of cetuximab-IRDye 800CW,cetuximab-Alexafluor488, or Cetuximab-IRDye 700DX conjugated with orwithout IRDye 800CW or Alexafluor488 in BxPC3 cells. The results areshown as a percent of PIT activity compared to Cetuximab-IRDye 700DX(control).

FIG. 24A shows the effect of pre-exposure of cetuximab-IRDye 700DXconjugate, cetuximab-IRDye 680RD conjugate, and cetuximab-IRDye700+IRDye 680RD dual conjugate to white light or green light on solubleaggregate formation.

FIG. 24B shows the effect of pre-exposure of cetuximab-IRDye 700DXconjugate, cetuximab-IRDye 680RD conjugate, and cetuximab-IRDye700+IRDye 680RD dual conjugate to white light or green light onfluorescence normalized to monomer content.

DETAILED DESCRIPTION I. Photoimmunotherapy Methods

Provided herein are conjugates, compositions, combinations and methodsrelated to photoimmunotherapy (PIT). Photoimmunotherapy is a moleculartargeted therapy that utilizes a target-specific photosensitizer basedon phthalocyanine dye, such as a near infrared (NIR) phthalocyanine dye(e.g., IR700), conjugated to a targeting molecule targeting a protein,such as a cell surface protein on a cell in a disease or condition, suchas a cell in a tumor. For example, in some cases a phthalocyaninedye-conjugate used in photoimmunotherapy can include conjugation to amonoclonal antibody (mAb) targeting a cell surface protein receptor orreceptor expressed on a cell in the environment of a disease lesion,such as a tumor microenvironment, which can include tumor cells andother infiltrating cells. In some embodiments, activation of thedye-conjugate by irradiation with absorbing light, such as NIR light,excites the photosensitizer and results in cell killing, therebyreducing or eliminating the lesion (e.g., tumor) and treating thedisease or condition. In some cases, the use of light in the NIR rangeleads to deeper tissue penetration resulting in successful eradicationof tumors after only a single dose of external NIR light irradiation.

Generally, targeted phototoxicity appears to be primarily dependent onbinding of the dye-conjugate to the cell membrane via the specifictargeting molecule (e.g., a macromolecule, such as an antibody). Forexample, studies using an exemplary antibody-IR700 molecule indicatethat the conjugate must be bound to the cellular membrane to be active,and that cell killing does not require intracellular localization to beeffective (see, e.g., U.S. Pat. No. 8,524,239 and U.S. publishedapplication No. US20140120119). Photo-activation of the conjugate-boundcells results in rapid cell death and necrosis.

Typically, PIT results in cell death primarily of those cells to whichthe phthalocyanine-dye conjugate, such as IR700-antibody conjugate,binds after the cells are irradiated with NIR, while cells that do notexpress the cell surface protein recognized by the targeting molecule(e.g., antibody) are not killed in significant numbers. Thus, becausethe therapy is targeted specifically to disease cells, such as cells ina tumor, its effects are highly selective to disease tissue compared tohealthy tissue or cells. For example, although a targetedphotosensitizer can be distributed throughout the body, it is onlyactive where intense light is applied, reducing the likelihood ofoff-target effects. This is in contrast to non-PIT-based methods inwhich the activity of similar therapeutic targeting molecules (e.g.,therapeutic antibodies) that are not conjugated to a photosensitizer(e.g., IR700) cannot be localized, thereby resulting in significantrisks of off-target side effects. In some embodiments, the phototoxicagent is a phthalocyanine dye-targeting molecule conjugate. In someembodiments, the phthalocyanine dye is IR700.

In some embodiments, the provided methods involve administering thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) in a dosage amount to achieve an exposure that is far lowerthan the exposure that would otherwise be required to achieve atherapeutic effect of the targeting molecule (e.g., antibody) that isnot conjugated to a phthalocyanine dye. Since PIT requires binding ofthe conjugated targeting molecule (e.g., antibody) to a cell surfaceprotein to mediate cell killing, it was believed that receptor occupancywould be a factor that would be correlated with the extent of PITactivity. Thus, it was believed that a similar, or even higher, systemicexposure of the conjugate would be necessary to ensure exposure at thesite of the lesion (e.g., tumor) to achieve sufficient cell killing aswould be required to achieve a clinically acceptable therapeutic effectof the targeting molecule (e.g., antibody) that was not so conjugated(i.e. not conjugated to the photosensitizer, e.g., IR700). For example,the dose of Erbitux® (cetuximab) approved by the Food and DrugAdministration (FDA) to treat Head and Neck cancer is an initial dose of400 mg/m² followed by a weekly administration of 250 mg/m². The averagesystemic exposure (AUC, e.g., AUC_(0-inf) or AUC[0-inf]) in a samplepatient population resulting from single dosage administration ofErbitux at 400 mg/m² is about 24,620 μg*h/mL and the total systemicexposure for a one (1)-month treatment (initial dose of 400 mg/m²followed by weekly administration of 250 mg/m²) is estimated to be about60,056 μg*h/mL (cumulative systemic exposures of the four weekly dosesin 1 month, 1×24,620+3×11,812 μg*h/mL) (Fracasso et al. (2007) Clin.Cancer. Res., 13:986).

It is found herein, however, that cell killing by PIT could be observedfollowing single dosage administration of an amount of cetuximab-IR700conjugate that achieved a mean systemic exposure of only about 1810μg/mL*h (AUC_(0-inf)) or 770+/−47.5 (AUC₀₋₂₄) in a sample patientpopulation. This amount is up to or about 13.6-fold than the systemicexposure observed following the clinical therapeutic doses of Erbitrux®at 400 mg/m² described above. Further, as shown in Example 2, theresults showed that the average systemic exposure (AUC₀₋₂₄) in a samplepatient population even at a higher dose of 640 mg/m² was approximately15% of the AUC for 400 mg/m² Erbitux® (3,690 vs. 24,740 μg/mL*h,respectively). The results further showed that in patients with head andneck cancer (which included patients that had failed other treatments),there was a 100% objective response rate (ORR) to the single dosagetreatment (all patients exhibited a complete or partial response to thetreatment), demonstrating a rapid and robust response that wassurprising considering the low systemic exposure. By comparison, the ORRof subjects treated with Erbitux® monotherapy is usually about 15% orbelow, even with the multiple doses required for continuous exposure.

Further, most treatments with Erbitux® involve combination therapy withone or more chemotherapeutic or other anti-cancer treatments. As anexample, in one reported study, the increase of ORR by cetuximab incombination with chemotherapy compared to chemotherapy alone is onlyabout 16%, which is similar to the ORR provided by cetuximab monotherapy(see, e.g., Specenier and Vermorken (2013) Biologics, 7:77-90).

Thus, the provided methods are based on observations that the potency ofa phthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) for mediating PIT and cell killing is sufficiently high evenwhen the systemic exposure of the conjugate is low. Thus, the providedmethods include administration of the conjugate at doses that achieve asystemic exposure that is a fraction of the exposure of a clinical dosein humans of the corresponding therapeutic targeting molecule (e.g.,therapeutic antibody) when it is not so conjugated to a phthalocyaninedye (e.g., IR700). In some embodiments, the clinical dose in humans of atherapeutic molecule is a dose as described at the label approved forcommercialization by regulatory agencies (e.g. FDA, EMA, PDMA). In someembodiments, the low systemic exposure achieved by such doses of thephthalocyanine dye-targeting molecule conjugate means that the systemicexposure of the targeting molecule is not sufficiently high to exhibitpharmacologic activity, unless PIT is induced by exposure of theconjugate to light. Since irradiation with a dose of light can belocalized to the disease lesion (e.g., tumor), the provided methods canachieve selective cell killing only of the disease lesion (e.g., tumor)while avoiding unwanted or undesired off-target activity.

Besides the potency of the response provided by the PIT methods providedherein, the provided methods also are particularly advantageous fortreating disease or conditions (e.g., tumors) in which existingtherapies, such as existing antibody therapies, are prone to result inadverse off-target side effects. For example, in some cases, it isdifficult to achieve strong anti-cancer activity in the range of safetyusing therapies involving immune modulating agents. In some cases, thisis because such immune modulating agents inhibit (or enhance) mechanismsthat are the same mechanisms in which our body uses to fightautoimmunity. As a result, in many cases, dosing with immune modulatingagents can result in significant therapeutic side effects. For safety ofmany immune modulating agents (and other therapeutic antibodies), theacceptable doses compromise therapeutic efficacy, must be administeredin a dosage cycle to achieve or maintain a continuous threshold systemicexposure and/or must be administered in combination with otherchemotherapeutic or anti-cancer agents that risk even greater adverseside effects. The provided methods solve these problems becausePIT-based conjugates containing therapeutic targeting molecules,including immune modulating agents and other anti-tumor antibodymolecules, can be administered at doses that avoid or minimize highsystemic exposure, while also permitting selective cell killing at thesite of the disease or lesion. Thus, the advantage of low dosing and/orlow systemic exposure is significant for achieving safety.

In some embodiments, the fluorescent properties of the phthalocyaninedye also permits monitoring of the PIT therapy using any of a number ofimaging or other method capable of detecting fluorescent signal. In someembodiments, evaluation of fluorescence, such as by imaging, can be usedto monitor the upload or presence of the conjugate at the lesion (e.g.,tumor) prior to PIT. In some embodiments, evaluation of fluorescence,such as by imaging, can be used to illuminate the lesion (e.g., tumor)to ensure PIT is directed at the site of the lesion (e.g., tumor). Insome embodiments, evaluation of fluorescence can use used in thesurgical setting where the margins of the lesion (e.g., tumor) can bevisualized with fluorescence and then residual cancer cells in themargins can be killed with PIT.

In some embodiments, the conjugate further contains an additionalfluorescent dye in addition to the phthalocyanine dye (e.g., IR700). Forexample, in some cases, IR700 is not among the most ideal dyes forimaging because, for example, it exhibits one or more spectralproperties that may not be as suitable to ensure specific and efficientfluorescent labeling. In some aspects, other fluorophores commonly usedto label proteins can exhibit a higher fluorescent quantum yield, alarger Stokes shift, a larger extinction coefficient at the excitationwavelength and/or a longer wavelength for deeper tissue penetration.Thus, in some embodiments, a dual-label conjugate is provided in which atargeting molecule (e.g., antibody or antigen-binding antibody fragment)is conjugated to a first dye that is a phthalocyanine dye (e.g., IR700)and also is conjugated to another second fluorescent dye that exhibitsone or more properties from among a higher quantum yield, larger Stokesshift, larger extinction coefficient and/or longer wavelength that isimproved or better as compared to the first dye. In some embodiments,the dual-label conjugate can be used to both monitor PIT therapy asdescribed above and also to treat with PIT by activating thephthalocyanine dye, such as activating IR700. In some embodiments, thefirst and second dye are selected to minimize energy transfer betweenthem, for example, the first and second dye are selected to avoid orminimize overlapping emission and absorption spectra.

In some embodiments, also provided herein are combination therapies foruse in concert with photoimmunotherapy for treating a disease orcondition in a subject. In some embodiments, the combination therapy canbe used in methods for treating a tumor or cancer. In some embodiments,the combination of photoimmunotherapy and administration of anadditional therapeutic agent, such as an immune modulating agent,anti-cancer agent or other agent, can increase the efficacy of treatingthe tumor, which, in some cases, can increase the therapeutic outcome orsurvival of the treated subject.

In some aspects, the provided combination therapy methods exploit thecytotoxic killing and/or lysis effects induced by PIT to enhancetherapeutic outcomes in connection with tumor therapy. In particularaspects, one or more additional therapeutic agents can be administeredto a subject having a tumor prior to completing PIT, which occurs bylight irradiation to activate the phthalocyanine dye-conjugate. In someembodiments, the prior administration of the additional therapeuticagent can prime the tumor microenvironment to be more responsive to thePIT or to the additional therapeutic agent following the subsequentirradiation of the tumor.

For example, in one embodiment, an additional therapeutic agent can bean immune modulating agent, which, in some aspects, is administeredprior to irradiation in order to enhance the immune response toPIT-induced tumor-associated agents released from lysed cells. Inanother example, an additional therapeutic agent can be an anti-canceragent, which, in some aspects, is administered prior to irradiation toincrease systemic availability of the anti-cancer agent to enhancedelivery or uptake of the anti-cancer agent into the tumor area inresponse to changes in tumor permeability induced by PIT. In someembodiments, the enhanced therapeutic outcome from the combinationtherapy can result in an increased reduction in tumor size (e.g., tumorvolume or weight) or an increased or longer survival of the subjectcompared to methods involving treatment with either therapy alone. Insome embodiments, the therapeutic effect of the combination therapy canbe synergistic compared to treatment methods involving treatment withthe phthalocyanine dye-conjugate/PIT alone or treatments involving theadditional therapeutic agent alone, such as treatments with only theimmune modulating agent or only the anti-cancer agent.

A. Conjugates Containing a Phthalocyanine Dye and Targeting Molecule

The methods, compositions and combinations provided herein include aconjugate containing a photosensitizer, such as a phthalocyanine dye,for example IR700, and a targeting molecule (e.g., antibody or anantigen binding fragment of an antibody) that binds to a cell surfaceprotein. In some embodiments, binding of the targeting molecule that isconjugated to the photosensitizer, such as a phthalocyanine dye (e.g.,IR700), to the cell surface protein permits the targeting of theconjugate to cells involved in a disease or condition, such as a tumoror cancer, infection, inflammatory disease or condition, neuronaldisease or condition or other diseases or conditions. In someembodiments, the targeted cells (e.g., cells expressing the cell surfaceprotein capable of being bound by the targeting molecule) are present inthe microenvironment of a lesion associated with the disease orcondition, for example, the cells are present in a tumormicroenvironment. In some embodiments, cell targeting increases theefficacy of PIT induced upon local irradiation of the lesion (e.g.,tumor) of the subject at a wavelength that is absorbed by thephthalocyanine dye (e.g., a near-infrared (NIR) wavelength), since cellkilling is selective to those cells in which the dye-targeting moleculeconjugate is bound.

In some embodiments, the phthalocyanine dye conjugates for use in thecombination therapy provided herein include a dye molecule conjugated toa targeting molecule via a linker group. In one aspect, the conjugate isof Formula I:

A-[(L)_(n)-D]_(p)   (I)

wherein:

-   -   A is a targeting molecule that can bind to cells or tissues;    -   L is an independently selected linker for each p;    -   n is 1 or 2;    -   D is an independently selected hydrophilic phthalocyanine dye        for each p; and    -   p is independently 1, 2, 3, 4, 5 or greater than 5, such as up        to 1000. For example, p can be 1 to 1000, such as generally 1 to        10 or 2 to 5.

In some embodiments, the phthalocyanine dye conjugate is produced by amethod or process in which the phthalocyanine dye-targeting moleculeconjugate, such as an IR700-targeting molecule (e.g., IR700-antibody)conjugate, is prepared under light-protected conditions. In someembodiments, the method includes 1) preparing or providing aphthalocyanine dye and a targeting molecule; 2) contacting the targetingmolecule and phthalocyanine dye under conditions to generate theconjugate with minimal exposure of the dye; and 3) formulating,purifying and/or isolating the conjugate to produce a compositioncontaining the drug substance, where one or more of the steps, such asin some cases all of the steps, are performed with minimal exposure ofthe dye or the conjugate containing the dye to environmental light. Insome embodiments, the phthalocyanine dye-targeting molecule conjugate,such as an IR700-targeting molecule (e.g., IR700-antibody) conjugate, isa conjugate, or is prepared using methods for producing a conjugate, asdescribed in U.S. Application No. 62/206,774, which is incorporated byreference herein.

1. Phathalocyanine Dye

Phthalocyanines are a group of photosensitizer compounds having thephthalocyanine ring system. Phthalocyanines are azaporphyrins thatcontain four benzoindole groups connected by nitrogen bridges in a16-membered ring of alternating carbon and nitrogen atoms (i.e.,C₃₂H₁₆N₈) which form stable chelates with metal and metalloid cations.In these compounds, the ring center is occupied by a metal ion (either adiamagnetic or a paramagnetic ion) that may, depending on the ion, carryone or two ligands. In addition, the ring periphery may be eitherunsubstituted or substituted. The synthesis and use of a wide variety ofphthalocyanines in photodynamic therapy are described in InternationalPublication WO 2005/099689 and U.S. Pat. No. 7,005,518.

In some embodiments, phthalocyanines strongly absorb red or near IRradiation with absorption peaks falling between about 600 nm and 810 nm,which, in some cases, allow deep penetration of tissue by the light.Phthalocyanines are generally photostable. This photostability istypically advantageous in pigments and dyes and in many of the otherapplications of phthalocyanines.

In some embodiments, the phthalocyanine dye is water soluble andcontains a luminescent fluorophore moiety having at least oneaqueous-solubilizing moiety. In some embodiments, the aqueoussolubilizing moiety contains silicon. In some embodiments, thephthalocyanine dye has a core atom such as Si, Ge, Sn, or Al. In someembodiments, the phthalocyanine dye exists as a single core isomer,essentially free of other isomers. In some embodiments, thephthalocyanine dye contains a linker that has a reactive or activatablegroup, which is able to form a bond between the linker and targetingmolecule. In some embodiments, the phthalocyanine dye can be tailored tofluoresce at a particular wavelength.

In some embodiments, the phthalocyanine dye contains a linker, i.e., isa linker-phthalocyanine dye moiety (L-D). In some embodiments, thelinker contains a reactive group. In some embodiments, thephthalocyanine dye is of Formula Ia:

wherein

L is selected from a direct link, or a covalent linkage;

Q is a reactive group or an activatable group that can be part of thelinker L, and is any group that can react to form a bond between L andthe targeting molecule A;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹, if present, are each independentlyselected from hydrogen, optionally substituted alkyl, optionallysubstituted alkanoyl, optionally substituted alkoxycarbonyl, optionallysubstituted alkylcarbamoyl, or a chelating ligand, wherein at least oneof R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachfunctional groups that can be independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino or optionally substituted alkoxy;

or in an alternative embodiment, at least one of i) R¹³ and R¹⁴, and thecarbons to which they are attached, or ii) R¹⁷ and R¹⁸, and the carbonsto which they are attached, or iii) R²¹ and R²², and the carbons towhich they are attached, join to form a fused ring; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

In some embodiments, L is a covalent linkage. In some embodiments, thecovalent linkage is linear or branched, cyclic or heterocyclic,saturated or unsaturated, having 1-60 atoms, such as 1-45 atoms or 1-25atoms. In some cases, such atoms can be selected from C, N, P, O, and S.In some embodiments, L can have additional hydrogen atoms to fillvalences (in addition to the 1-60 atoms). Generally, the linkagecontains any combination of ether, thioether, amine, ester, carbamate,urea, thiourea, oxy or amide bonds; or single, double, triple oraromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur,nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; oraromatic or heteroaromatic bonds.

In some embodiments, L is of the formula —R¹—Y—X¹—Y¹—, wherein R¹ is abivalent radical or direct link; Y and Y¹ are each independentlyselected from t a direct link, oxygen, an optionally substitutednitrogen, or sulfur; and X¹ is selected from t a direct link and C₁-C₁₀alkylene optionally interrupted by an atom. Bivalent radicals include,but are not limited to, optionally substituted alkylene, optionallysubstituted alkyleneoxycarbonyl, optionally substitutedalkylenecarbamoyl, optionally substituted alkylenesulfonyl, andoptionally substituted arylene.

Exemplary R¹ substituents include, but are not limited to, optionallysubstituted alkylene, optionally substituted alkyleneoxycarbonyl,optionally substituted alkylenecarbamoyl, optionally substitutedalkylenesulfonyl, optionally substituted alkylenesulfonylcarbamoyl,optionally substituted arylene, optionally substituted arylenesulfonyl,optionally substituted aryleneoxycarbonyl, optionally substitutedarylenecarbamoyl, optionally substituted arylenesulfonylcarbamoyl,optionally substituted carboxyalkyl, optionally substituted carbamoyl,optionally substituted carbonyl, optionally substituted heteroarylene,optionally substituted heteroaryleneoxycarbonyl, optionally substitutedheteroarylenecarbamoyl, optionally substitutedheteroarylenesulfonylcarbamoyl, optionally substitutedsulfonylcarbamoyl, optionally substituted thiocarbonyl, a optionallysubstituted sulfonyl, and optionally substituted sulfinyl.

In some embodiments, Q contains a reactive group for optional attachmentto a material, such as a targeting molecule. As used herein, the term“reactive group” means a moiety on the compound that is capable ofchemically reacting with the functional group on a different material(e.g., targeting molecule) to form a linkage, such as a covalentlinkage. Typically, the reactive group is an electrophile or nucleophilethat can form a covalent linkage through exposure to the correspondingfunctional group that is a nucleophile or electrophile, respectively.Alternatively, the reactive group is a photoactivatable group, andbecomes chemically reactive only after illumination with light of anappropriate wavelength. Typically, the conjugation reaction between thereactive dye and the targeting molecule to be conjugated results in oneor more atoms of the reactive group Q incorporated into a new linkageattaching the dye to the conjugated targeting molecule.

In some embodiments, Q contains a reactive group that is reactive with acarboxyl group, an amine, or a thiol group on the targeting molecule.Suitable reactive groups include, but are not limited to, an activatedester, an acyl halide, an alkyl halide, an anhydride, a carboxylic acid,a carbodiimide, a carbonate, a carbamate, a haloacetamide (e.g.,iodoacetamide), an isocyanate, an isothiocyanate, a maleimide, an NHSester, a phosphoramidite, a platinum complex, a sulfonate ester and athiocyanate for optional attachment to the targeting molecule. In someembodiments, the reactive groups are reactive with a carboxyl group, anamine, or a thiol group on a targeting molecule. In some embodiments,the reactive group is a sulfhydryl-reactive chemical group such asmaleimide, haloacetyl, and pyridyl disulfide. In some embodiments, thereactive group is amine-reactive. In some embodiments, the reactivegroup is an NHS ester.

In some embodiments, R², R³, R⁷, and R⁸ are each optionally substitutedalkyl such as optionally substituted methyl, ethyl, or isopropyl groups.

In some embodiments, at least one of R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹contains a water soluble group. For example, the alkyl portion of R⁴,R⁵, R⁶, R⁹, R¹⁰, and R¹¹ is substituted with a water solublesubstituent. As used herein, “water soluble group” refers to a groupcomprising one or more polar and/or ionic substituents that improves thesolubility of the overall molecule in aqueous media. In some cases, atleast two of R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ comprise water soluble groups.In other embodiments, three or more comprise water soluble groups. Watersoluble groups include, but are not limited to, a carboxylate (—CO₂ ⁻)group, a sulfonate (—SO₃ ⁻) group, a sulfonyl (—SO₂ ⁻) group, a sulfate(—SO₄ ⁻²) group, a hydroxyl (—OH) group, a phosphate (—OPO₃ ⁻) group, aphosphonate (—PO₃ ⁻²) group, an amine (—NH₂) group and an optionallysubstituted quaternized nitrogen with each having an optional counterion.

Suitable counter ions include, but are not limited to, sodium,potassium, calcium, ammonium, organic amino salt, or magnesium salt, ora similar salt. Preferably, the counter ion is a biologically acceptablecounter ion.

In some embodiments, the nitrogen atom(s) to which R⁴, R⁵, R⁶, R⁹, R¹⁰,and R¹¹ are attached can be trivalent or tetravalent.

In some embodiments, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹,R²² and R²³ are each hydrogen.

In some embodiments, X² and X³ are each independently selected fromC₁-C₁₀ alkylene optionally interrupted by an atom. In some embodiments,the nitrogens appended to X² and/or X³ can be optionally quaternized.

In some embodiments, the phthalocyanine dye is of Formula Ib:

wherein

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

and

R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹⁶, R¹⁷, R¹⁸, R¹⁹, X² and X³are as defined herein.

In some embodiments, the reactive group is an NHS ester. In someembodiments, the reactivity of the NHS ester can be adjusted by varyingthe length of the alkylene group of X⁴, between the NHS ester andcarbamate functionality. In some embodiments, the length of the alkylenegroup of X⁴ between the NHS ester and the carbamate functionality isinversely proportional to the NHS ester reactivity. In some embodiments,X⁴ is C₅-alkylene. In other embodiments, X⁴ is C₃-alkylene. In someembodiments, X¹ is C₆-alkylene. In other embodiments, X¹ is C₃-alkylene.

In some embodiments, the phthalocyanine dye has an overall electroniccharge of zero. This charge neutrality can in certain instances byobtained with one or more optional counterions, or quaternizednitrogens.

In some embodiments, the phthalocyanine dye has sufficient solubility inaqueous solutions that once it is attached to a soluble targetingmolecule, the targeting molecule retains its solubility. In someembodiments, the dye also is soluble in organic media (e.g., DMSO orDMF).

In some embodiments, the phthalocyanine dye has a maximum lightabsorption in the near infrared (NIR range). In some embodiments, thephthalocyanine dye has a maximum light absorption wavelength between 400nm and 900 nm, such as between 600 nm and 850 nm, such as between 680 nmand 850 nm, for example at approximately 690 nm±50 nm or 690±20 nm. Insome embodiments, the phthalocyanine dye can be excited efficiently bycommercially available laser diodes that emit light at thesewavelengths.

In some embodiments, the phthalocyanine dye containing the reactivegroup is IR700 NHS ester, such as IRDye 700DX NHS ester (L₁-Ccor929-70010, 929-70011). Thus, in some embodiments, the dye is a compoundhaving the following formula:

For purposes herein, the term “IR700,” “IRDye 700DX,” or variationsthereof refer to the above formula when the dye is conjugated to atargeting molecule via its reactive group. Generally, IR700 has severalfavorable chemical properties. Amino-reactive IR700 is a relativelyhydrophilic dye and can be covalently conjugated with an antibody usingthe NHS ester of IR700. Typically, IR700 also has more than 5-foldhigher extinction coefficient (2.1×10⁵M⁻¹cm⁻¹ at the absorption maximumof 689 nm), than conventional photosensitizers such as thehematoporphyrin derivative Photofrin® (1.2×10³M⁻¹cm⁻¹ at 630 nm),meta-tetrahydroxyphenylchlorin; Foscan® (2.2×10⁴ M⁻¹cm⁻¹ at 652 nm), andmono-L-aspartylchlorin e6; NPe6/Laserphyrin® (4.0×10⁴M⁻¹cm⁻¹ at 654 nm).

The phthalocyanine dyes described herein can be made with commerciallyavailable starting material. The core structure is synthesized bycondensation of two or more different diiminoisoindolines. Syntheticstrategies using different dinitriles or diiminoisoindolines can lead tovarious degrees of substitution of the phthalocyanine and/ordistribution of regioisomers. Exemplary synthetic schemes for generatingthe dyes are described in U.S. Pat. No. 7,005,518.

In some embodiments, in any of the methods provided herein, thetargeting molecule (e.g. antibody) is linked directly or indirectly tothe phthalocyanine dye (e.g. IR700). In some embodiments, the targetingmolecule (e.g. antibody) is linked, directly or indirectly, to thephthalocyanine dye (e.g. IR700) via a covalent bond or a non-covalentinteraction. In some embodiments, the covalent or non-covalentinteractions or linkage is direct or indirect. In some embodiments, theattachment includes an indirect link, such as through a linker (e.g.such as any of the exemplary linkers described above), binding moiety ordomain or reactive group. In some embodiments, the linkage includes adirect interaction between the targeting molecule and a phthalocyaninedye (e.g., IR700). In other embodiments, one or both of the targetingmolecule and the phthalocyanine dye are linked to one or more linkers,and the interaction is indirect, e.g., between a linker attached to oneof the molecules and another molecule, or between two linkers, eachattached to the targeting molecule or the phthalocyanine dye.

In some embodiments, the phathalocyanine dye is non-covalently linked toor associated with the targeting molecule. For example, thephathalocyanine dye forms a complex with the targeting molecule via anon-covalent interaction. In some embodiments, the phthalocyanine dye(e.g. IR700) contains a moiety or domain capable of non-covalentlyinteracting with an attachment group of the targeting molecule. In someembodiments, the method includes incubating or binding thephthalocyanine dye (e.g. IR700) with the targeting molecule (e.g.antibody) to form a non-covalent interaction between the dye and thetargeting molecule. In some examples, the non-covalent interactionbetween the targeting molecule and the phthalocyanine dye include, forexample, electrostatic interactions, van der Waals force, hydrophobicinteractions, π-effects, ionic interactions, hydrogen bonding, halogenbonding and/or combinations thereof, or any interactions that depend onone or more of the forces. In some embodiments, the targeting moleculeand the phthalocyanine dye are linked using or using interactions thatmimic non-covalent molecular interactions such as, for example,ligand-receptor interaction, antibody-antigen interaction, avidin-biotininteraction, streptavidin-biotin interaction, histidine-divalent metalion interaction (e.g., Ni, Co, Cu, Fe), interactions betweenmultimerization (e.g., dimerization) domains, glutathione S-transferase(GST)-glutathione interaction and/or any combination thereof.

In some embodiments, a non-covalent interaction moiety or domain isattached to or is a part of the targeting molecule, and forms anon-covalent interaction, e.g. a complex, with the phthalocyanine dye(e.g. IR700). In other embodiments, non-covalent interaction molecule ordomain is attached to or is a part of the phthalocyanine dye molecule,and forms a non-covalent interaction e.g. a complex, with the targetingmolecule. In some embodiments, the method includes incubating orcontacting a targeting molecule conjugated to biotin (e.g.antibody-biotin, such as a cetuximab-biotin) and the phthalocyanine dyeconjugated to an avidin or analog thereof or a streptavidin or analogthereof, including monomeric forms thereof (e.g. monomeric avidin-IR700or monomeric streptavidin-IR700). By virtue of the non-covalentinteraction between avidin, streptavidin or analogs thereof and biotin,in some embodiments, the phthalocyanine dye (e.g. IR700) forms anon-covalent complex with the targeting molecule.

2. Targeting Molecule

In some embodiments, the phthalocyanine dye is conjugated to a targetingmolecule via a reactive group of the dye molecule. In some embodiments,the targeting molecule is one that is able to target the conjugate to acell or pathogen, for example, by binding to a cell surface molecule(e.g. cell surface receptor) on the cell or pathogen. In someembodiments, the targeting molecule, e.g., a macromolecule, canselectively bind to a desired cell type, cells with a particularphenotype, or cells displaying one or more cell surface markers orantigens. In some cases, the targeting molecule binds to a cell that isa cancer cell, a tumor cell, an inflammatory cell, an immune cell, aneuron, a stem cell, a proliferating cell, or a cell in a hyperplasia.In some cases, the targeting molecule binds to a pathogen or a pathogeninfected cell. In some embodiments, the cell is an inflammatory cell,such a leukocyte, for example, a neutrophil, an eosinophil, a basophil,a lymphocyte, or a monocyte. In some embodiments, the cell is an immunecell, such as a T cell, a B cell, a Natural Killer (NK) cell, adendritic cell, a macrophage or a neutrophil. In some embodiments, thecell is a neuron that is a peripheral nervous system neuron or a centralnervous system neuron, such as a nociceptor, for example, thermalnociceptors, mechanical nociceptors, chemical nociceptors or polymodalnociceptors. In some cases, the targeting molecule binds to a pathogenor a pathogenic cell, such as a virus, bacterium, fungus, biofilm orother prokaryotic cell system. In some embodiments, the targetingmolecule binds to a pathogen that is a gram-negative or gram-positivebacterium.

In some embodiments, the targeting molecule (e.g., antibody) of thephthalocyanine dye conjugate bind to a protein on the surface of a cellor cells present in a microenvironment of a lesion that is associatedwith or present as a result of a disease or condition. For example, insome embodiments, the conjugate binds to a protein on the surface of acell or cells present in a tumor microenvironment associated with orpresent in a tumor. In some embodiments, the conjugate binds to aprotein present the extracellular matrix in the microenvironment of thetumor.

As used herein, a “cell present in the microenvironment of a lesion”refers to any cell present in the cellular environment associated with alesion, a disease or a disorder, such as any cell present in orimmediately adjacent to a tumor, such as cells present in a tumormicroenvironment, or the extracellular matrix in the tumormicroenvironment.

As used herein, a “cell present in a tumor microenvironment” refers toany cell present in the cellular environment in which the tumor exists,such as any cell present in or immediately adjacent to the tumor,including the proliferating tumor cells (e.g., cancer cells), the tumorstroma, blood vessels, infiltrating inflammatory cells (e.g., immunecells) and a variety of associated tissue cells (e.g., fibroblasts).Thus, it is understood that reference to the tumor refers not only tothe tumor cells, which can include malignant or cancer cells, but alsoto other cells present in the tumor microenvironment that regulate thegrowth of the tumor, including immune cells. In some cases, immune cellspresent in a tumor microenvironment can include T lymphocytes, includingregulatory T lymphocytes (Treg), dendritic cells, natural killer (NK)cells, B cells, macrophages and other immune cells (Whiteside (2008)Oncogene, 27:5904-5912). It is recognized that, in some aspects, manynon-cancerous cells present in and around the tumor can regulate theproliferation, angiogenesis, invasion and/or metastasis of tumor cells,thereby promoting the growth of the tumor. Thus, in some cases,targeting such non-cancerous cells, such as immune cells (e.g., T cells,such as regulatory T cells), present in a tumor can be an effectivetherapy for killing a tumor by PIT.

Generally, cancerous cells contain antigens associated with a tumor thatshould be recognized by the immune system. Typically, in an activeimmune system, immune cells, such as cytotoxic T cells, attack anderadicate these cancerous cells. Under normal physiological conditions,the T cell-mediated immune response is initiated by antigen recognitionby the T cell receptor (TCR) and is regulated by a balance ofco-stimulatory and inhibitory signals (e.g., immune checkpointproteins). In particular, CD4+ and CD8+ T cells expressing a TCR canbecome activated upon recognition of antigenic peptides presented onantigen-presenting cells on major histocompatibility complex (MHC) classI or class II molecules, respectively. In some aspects, activated CD8+cells, or cytotoxic T cells, can kill tumor cells expressing theantigen, which can be helped by the presence of CD4+ T cells.

In the case of tumors, however, the tumor microenvironment hasmechanisms to suppress the immune system, thereby evading immunerecognition and preventing or reducing killing of tumor cells. Forexample, in some cases, immune checkpoint proteins can be dysregulatedin tumors, thereby resulting in a suppression of the immune response inthe tumor microenvironment as a mechanism of evading the immune system.In some cases, tumor-infiltrating lymphocytes can include Tregs (e.g.,CD4+CD25+ T cells), which are cells that are capable of suppressingproliferation of other T cells in the microenvironment (Whiteside, TL(2008) Oncogene, 27:5904-5912). In some cases, other mechanisms can actto inhibit access of immune cells to tumor antigens, thereby alsocontributing to the tumors ability to evade the immune system.

In some embodiments, the targeting molecule is a targeting molecule thatbinds to a cell surface protein on a tumor or cancer cell. In someembodiments, the targeting molecule binds to a cell surface protein onan immune cell or other non-cancerous cell present in a tumormicroenvironment. In some embodiments, the targeting molecule binds to acell surface protein on the surface of a T lymphocyte, such as a Treg, adendritic cell, a natural killer (NK) cell, a B cell, a macrophage orother immune cell that is present in a tumor microenvironment. In somecases, the tumor or cancer is located at the head and neck, breast,liver, colon, ovary, prostate, pancreas, brain, cervix, bone, skin, eye,bladder, stomach, esophagus, peritoneum, or lung.

Exemplary of targeting molecules, such as targeting molecules thattarget a tumor or cancer, include, but are not limited to, any asdescribed in published international PCT appl. Nos. WO2014120974,WO2014176284, WO2015042325, U.S. Pat. No. 8,524,239 or U.S. patentpublication No. US20140120119.

Exemplary targeting molecules include, but are not limited to, aprotein, a glycoprotein, an antibody, an antibody fragment, an antigen,an antigen binding fragment, a peptide, a polypeptide, a small molecule,a polymeric synthetic molecule, a polymeric nanoparticle, a liposome, anenzyme substrate, a hormone, a neurotransmitter, a cell metabolite, aviral particle, a viral capsid, a viral nanoparticle, a bacterialparticle, a marker, a cell, a hapten, an avidin, a streptavidin, amonomeric streptavidin, a biotin, a carbohydrate, an oligosaccharide, apolysaccharide, a nucleic acid, a deoxy nucleic acid, a fragment of DNA,a fragment of RNA, nucleotide triphosphates, acyclo terminatortriphosphates, or PNA.

In some embodiments, the targeting molecule is an amino acid, peptide,protein, tyramine, polysaccharide, ion-complexing moiety, nucleoside,nucleotide, oligonucleotide, psoralen, drug, hormone, lipid, lipidassembly, polymer, polymeric microparticle, a biological cell, or virus.In some embodiments, the targeting molecule is an antigen, steroid,vitamin, drug, metabolite, toxin, environmental pollutant, nucleic acidpolymer, carbohydrate, lipid, or glass, plastic or other non-biologicalpolymer. In some embodiments, the targeting molecules is a cell,cellular system, cellular fragment, or subcellular particle, e.g., avirus particle, bacterial particle, virus component, biological cell(such as animal cell, plant cell, bacteria, yeast, or protist), orcellular component. In some embodiments, reactive dyes may labelfunctional groups at the cell surface, in cell membranes, organelles, orcytoplasm.

In some embodiments, the targeting molecule targets or binds to anantigen, such as any structural substance that serves as a targetcapable of being bound by the targeting molecule. In some embodiments,the antigen is or is comprised as part of a cell surface molecule, suchas a protein, e.g., a receptor, that is expressed on a cell surface. Insome embodiments, for example, the antigen is or is comprised as part ofa molecule expressed on the surface of a cell present in a tumor,including any cell present in the tumor microenvironment. Examples ofcell surface molecules include, but are not limited to, an antigen,peptides, lipids, polysaccharides, carbohydrate, or nucleic acidscontaining antigenic determinants, such as those recognized by an immunecell. In some examples, an antigen includes a tumor-specific peptide(such as one found on the surface of a cancer cell) or immunogenicfragment thereof. In some embodiments, the targeting molecule is anantibody or an antigen-binding antibody fragment thereof.

In some embodiments, the cell surface molecule can be ACTHR, endothelialcell Anxa-1, aminopetidase N, anti-IL-6R, alpha-4-integrin,alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha-fetoprotein(AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2,B-cell receptor BB1, BB2, BB4, calcitonin receptor, cancer antigen 125(CA 125), CCK1, CCK2, CD S, CD10, CD11a, CD13, CD14, CD19, CD20, CD22,CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15,CD34, CD44, CD206, CD271, CEA (CarcinoEmbryonic Antigen), CGRP,chemokine receptors, cell-surface annexin-1, cell-surface plectin-1,Cripto-1, CRLR, CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR,EGFRvIII, EMR1, Endosialin, EP2, EP4, EpCAM, EphA2, ET receptors,Fibronectin, Fibronectin ED-B, FGFR, frizzled receptors, GAGE1, GAGE2,GAGE3, GAGE4, GAGE5, GAGE6, GLP-1 receptor, G-protein coupled receptorsof the Family A (Rhodopsin-like), G-protein coupled receptors of theFamily B (Secretin receptor-like) like), G-protein coupled receptors ofthe Family C (Metabotropic Glutamate Receptor-like), GD2, GP100, GP120,Glypican-3, hemagglutinin, Heparin sulfates, HER1, HER2, HER3, HER4,HMFG, HPV 16/18 and E6/E7 antigens, hTERT, IL11-R, IL-13R, ITGAM,Kalikrien-9, Lewis Y, LH receptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2,MAGE 3, MAGE 4, MART 1, MC1R, Mesothelin, MUC1, MUC16, Neu (cell-surfaceNucleolin), Neprilysin, Neuropilin-1, Neuropilin-2, NG2, NK1, NK2, NK3,NMB-R, Notch-1, NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2,NTR3, p32 (p32/gC1q-R/HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR,PDFG receptors, PDT, Protease-cleaved collagen IV, proteinase 3,prohibitin, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family(e.g., P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patched, RET receptor,plexins, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P,TEMs, T-cell CD3 Receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A,Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6,TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGFreceptors (VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, and VEGF-3 or FLT-4),voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, orY5.

In some embodiments, the targeting molecule is a binding partner, suchas a ligand, capable of binding to a cell surface molecule, such as acell surface protein, e.g., a cell surface receptor. In someembodiments, the targeting molecule is selected from adrenocorticotropichormone (ACTH), angiotensin II, atrial natriuretic factor (ANF),bombesin, bradykinin, brain derived neurotropihic factor (BDNF), bonemorphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6),bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1(BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor(CNTF), CCL1-CCL28, CXCL1-CXCL17, XCL1, XCL2, CX3CL1, cripto 1 bindingpeptide, vascular endothelial cell growth factor (VEGF), epidermalgrowth factor (EGF), endothelin 1, endothelin 1/3, FAS-ligand,fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2),fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5),fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7),fibroblast growth factor 1 (FGF-10), Flt-3, gastrin, gastrin releasingpeptide (GRP), granulocyte colony-stimulating factor (G-CSF),granulocyte macrophage stimulating factor (GM-CSF), glucagon likepeptide (GLP-1), hepatocyte growth factor (HGF), interferon alpha(IFN-α), interferon beta (IFN-b), interferon gamma (IFNg), insulin-likegrowth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2),interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9),interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12),interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17),interleukin 19 (IL-19), luteinizing hormone (LH), luteinizing-releasinghormone (LHRH), macrophage colony-stimulating factor (M-CSF), monocytechemotactic protein 1 (MCP-1), macrophage inflammatory protein 3a(MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growthfactor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4(NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylatecyclase activating peptide (PACAP), platelet derived growth factor AA(PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derivedgrowth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC),platelet derived growth factor DD (PDGF-DD), netrin-1 (NTN1), netrin-2(NTN2), netrin-4 (NTN4), netrin-G1 (NTNG1) and netrin-G2 (NTNG2), ephrinA1 (EFNA1), ephrin A2 (EFNA2), ephrin A3 (EFNA3), ephrin A4 (EFNA4),ephrin A5 (EFNAS), semaphorin 3A (SEMA3A), semaphorin 3B (SEMA3B),semaphorin 3C (SEMA3C), semaphorin 3D (SEMA3D), semaphorin 3F (SEMA3F),semaphorin 3G (SEMA3G), semaphorin 4A (SEMA4A), semaphorin 4B (SEMA4B),semaphorin 4C (SEMA4C), semaphorin 4D (SEMA4D), semaphorin 4F (SEMA4F),semaphorin 4G (SEMA4G), semaphorin 5A (SEMA5A), semaphorin 5B (SEMA5B),semaphorin 6A (SEMA6A), semaphorin 6B (SEMA6B), semaphorin 6D (SEMA6D),semaphorin 7A (SEMA7A), SLIT1, SLIT2, SLIT3, SLIT and NTRK-like family,member 1 (SLITRK1), SLIT and NTRK-like family, member 2 (SLITRK2), SLITand NTRK-like family, member 3 (SLITRK3), SLIT and NTRK-like family,member 4 (SLITRK4), SLIT and NTRK-like family, member 5 (SLITRK5), SLITand NTRK-like family, member 6 (SLITRK6), prostaglandin E2 (PGE2),RANTES, Somatostatin-14, Somatostatin-28, stem cell factor (SCF),stromal cell derived factor 1 (SDF-1), substance P, thyroid stimulatinghormone (TSH), transforming growth factor alpha (TGF-α), transforminggrowth factor beta (TGF-b), tumor necrosis factor alpha (TNF-α),thrombin, vasoactive intestinal peptide (VIP), Wnt1, Wnt2, Wnt2b/13,Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a,Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, or Wnt16, Sonichedgehog, Desert hedgehog, and Indian hedgehog, or is a binding fragmentthereof that is capable of binding to its cognate cell surface molecule,such as a cell surface protein, e.g., cell surface receptor.

In some embodiments, the targeting molecule can be an immune modulatingagent, which can bind to a cell surface molecule or protein on an immunecell to either suppress or activate the body's immune response. In someembodiments, binding of the immune modulating agent to the cell surfacemolecule or protein can stimulate an immune response to a tumor and/or apathogen, such as by inhibiting immune suppression or by enhancingimmunostimulation. In some embodiments, the cell surface molecule orprotein can be CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1), PD-L2 (CD273,B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137, TNFRSF9),CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTLA, CD160, VISTA (PD1homologue), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28, HHLA2(B7-H7), CD28H, CD155, CD226, TIGIT, CD96, Galectin 3, CD40, CD40L,CD70, LIGHT (TNFSF14), HVEM (TNFRSF14), B7-H3 (CD276), Ox40L (TNFSF4),CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GALS, NKG2A(CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, Butyrophilin family, CD48,CD244, Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide-relatedsequence A), MICB (MEW class I polypeptide-related sequence B), NKG2D,KIR family (Killer-cell immunoglobulin-like receptor, LILR family(Leukocyte immunoglobulin-like receptors, CD85, ILTs, LIRs), SIRPA(Signal regulatory protein alpha), CD47 (IAP), Neuropilin 1 (NRP-1), aVEGFR or VEGF. In some example, the targeting molecule is an antibody orantigen-binding fragment that is an immune modulating agent. In someembodiments, the immune modulating agent is an immune checkpointinhibitor.

In some embodiments, the cell surface molecule can be HER1/EGFR,HER2/ERBB2, CD20, CD25 (IL-2Ra receptor), CD33, CD52, CD133, CD206, CEA,CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA125),alpha-fetoprotein (AFP), Lewis Y, TAG72, Caprin-1, mesothelin, PDGFreceptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular endothelialgrowth factor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1and GM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCRcomplex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen,IgE, MUC-1, nuC242, PEM antigen, metalloproteinases, Ephrin receptor,Ephrin ligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor, or SK-1antigen.

In some embodiments, the targeting molecule is an antibody or anantigen-binding antibody fragment that specifically binds to an antigenthat is or is part of a cell surface molecule expressed on the surfaceof a cell. Included among such antibodies are antibodies orantigen-binding antibody fragments capable of binding to a cell surfacemolecule, such as a cell surface protein, e.g., cell surface receptor,described herein. In some cases, the antibody can bind to an antigen ofa protein expressed on a cell in a tumor, including a tumor-specificprotein.

In some embodiments, the targeting molecule binds to an antigen orprotein directly or indirectly. For example, in some embodiments, thetargeting molecule is a second binding molecule that binds to a firstbinding molecule which is capable of binding to the antigen or protein.For example, the targeting molecule is a secondary antibody, which bindsto a first binding molecule, e.g., a primary antibody, capable ofbinding the protein or antigen, e.g., a cell surface protein or a cellsurface receptor. Thus, in some embodiments, the dye is conjugated to asecondary antibody.

An “antibody” is a polypeptide ligand comprising at least a light chainand/or heavy chain immunoglobulin variable region that specificallyrecognizes and binds an epitope of an antigen. Generally, antibodies arecomposed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (V_(H)) region and the variable light(V_(L)) region. Together, the V_(H) region and the V_(L) region areresponsible for binding the antigen recognized by the antibody. The termantibody includes intact antibodies and antigen-binding antibodyfragments that exhibit antigen-binding, such as Fab fragments, Fab′fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). An scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as modified forms ofimmunoglobulins, chimeric antibodies, for example, humanized murineantibodies, and heteroconjugate antibodies, such as bispecificantibodies. See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes, or isotypes, which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, also known as “domains.” In combination, the heavy and the lightchain variable regions generally specifically bind the antigen. Lightand heavy chain variable regions may contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined (see, Kabat et al., Sequencesof Proteins of Immunological Interest, U.S. Department of Health andHuman Services, 1991, which is hereby incorporated by reference). TheKabat database is now maintained online. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species, such as humans. The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs are typically responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso generally identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. Antibodies with different specificities, such asdifferent combining sites for different antigens, have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

Among the provided antibodies are antibody fragments. An “antibodyfragment” refers to a molecule other than an intact antibody thatcomprises a portion of an intact antibody that binds the antigen towhich the intact antibody binds. Examples of antibody fragments includebut are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies;linear antibodies; single-chain antibody molecules (e.g., scFv); andmultispecific antibodies formed from antibody fragments. Other antibodyfragments or multispecific antibodies formed from antibody fragmentsinclude a multivalent scFv, a bispecific scFv or an scFv-CH3 dimer.Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs, which generally confer antigen binding, from anotherspecies, such as a murine antibody that specifically binds mesothelin.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In some embodiments,the CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they may be substantially identical to human immunoglobulin constantregions, such as at least about 85-90%, such as about 95% or moreidentical. Hence, parts of a humanized immunoglobulin, except possiblythe CDRs, are substantially identical to corresponding parts of naturalhuman immunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and CDRs from a humanimmunoglobulin. In some embodiments, the framework and the CDRs are fromthe same originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. Parts of a human immunoglobulinmay be substantially identical to corresponding parts of natural humanimmunoglobulin sequences.

“Specifically binds” refers to the ability of a molecule, such as anantibody or antigen-binding fragment, to specifically bind an antigen,such as a tumor-specific antigen, relative to binding to unrelatedproteins, such as non-tumor proteins, for example (3-actin. In someembodiments, a molecule, such as an antibody or fragment, including amolecule, such as an antibody or fragment, attached to a phthalocyaninedye molecule, specifically binds to a target, such as a cell surfaceprotein, with a binding constant that is at least 10³M⁻¹ greater, 10⁴M⁻¹greater or 10⁵M⁻¹ greater than a binding constant for other molecules ina sample or subject. In some embodiments, a molecule, such as anantibody or fragments thereof, has an equilibrium association constant(K_(A)) of greater than or equal to about 10⁶M⁻¹, greater than or equalto about 10⁷M⁻¹, greater than or equal to about 10⁸M⁻¹, or greater thanor equal to about 10⁹M⁻¹, 10¹⁰ M⁻¹, 10¹¹M⁻¹ or 10¹²M⁻¹. Antibodies alsocan be characterized by an equilibrium dissociation constant (K_(D)) of10⁻⁶ M, 10⁻⁷ M, 10⁻⁸M, 10⁻¹⁰ M, 10⁻¹¹M or 10⁻¹² M or lower. In someembodiments, an equilibrium dissociation constant (K_(D)) can be 1 nM orless. Affinity constants, such as K_(D) or K_(A), can be estimatedempirically or affinities can be determined comparatively, e.g. bycomparing the affinity of one antibody and another antibody for aparticular antigen. For example, such affinities can be readilydetermined using techniques known in the art, such as, for example, bycompetitive ELISA (enzyme-linked immunosorbent assay) or using asurface-plasmon resonance device, such as the Biacore T100 (availablefrom Biacore, Inc., Piscataway, N.J.), a radioimmunoassay usingradiolabeled target antigen, or by another method known to the skilledartisan.

In some embodiments, the phthalocyanine dye (e.g., IR700) is conjugatedto an antibody or an antigen-binding antibody fragment. For example, insome aspects, the phthalocyanine dye-targeting molecule conjugate is anIR700-antibody conjugate. Exemplary antibodies to which thephthalocyanine dye (e.g., IR700) can be conjugated to include, but arenot limited to, cetuximab, panitumumab, zalutumumab, nimotuzumab,trastuzumab, Ado-trastuzumab emtansine, Tositumomab (Bexxar®), Rituximab(Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin), Daclizumab(Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fab fragment,OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab (Avastin®),Afatinib, Axitinib, Bosutinib, Cabozantinib, Ceritinib, Crizotinib,Dabrafenib, Dasatinib, Erlotinib, Everolimus, Ibrutinib, Imatinib,Lapatinib, Lenvatinib, Nilotinib, Olaparib, Palbociclib, Pazopanib,Pertuzumab, Ramucirumab, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib,Temsirolimus, Trametinib, Vandetanib, Vemurafenib, Vismodegib,Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A,Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A, tremelimumab,IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166,dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MEDI6469, MEDI6383,MOXR0916, AMP-224, MSB0010718C, MEDI4736, PDR001, rHIgM12B7,Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab(BMS-986015, IPH2101), IPH2201, AGX-115, Emactuzumab, CC-90002 andMNRP1685A or an antibody-binding fragment thereof.

In some embodiments, the targeting molecule is a tissue-specific homingpeptide. For example, in some embodiments, the homing polypeptide cancontain the sequence of amino acids set forth in any of SEQ ID NOS:1-52. In some embodiments, the targeting molecule is an RGD polypeptide,such as an iRGD polypeptide, a Lyp-1 polypeptide, a cripto-1 bindingpolypeptide, a somatostatin receptor binding polypeptide, or aprohibitin binding polypeptide, a NGR polypeptide, or an iNGRpolypeptide.

In some embodiments, the targeting molecule is a viral particle, such asa virus-like particle, a viral-like nanoparticle, or a viral capsid. Insome embodiments, the targeting molecule is a viral-like nanoparticle.In some embodiments, the viral-like nanoparticle is assembled from L1capsid proteins. In some embodiments, the viral-like nanoparticle isassembled from a combination of L1 and L2 capsid proteins. In someembodiments, the targeting molecule and bind to and infect cells. Insome embodiments, the targeting molecule is one described inWO2015042325.

In some embodiments, a virus-like particle (VLP) refers to an organizedcapsid-like structure, such as roughly spherical or cylindrical inshape, that comprises self-assembling ordered arrays of L1 or L1 and L2capsomers and does not include a viral genome. In some embodiments,virus-like particles are morphologically and antigenically similar toauthentic virions, but they lack viral genetic material, such as viralnucleic acid, rendering the particles noninfectious. A VLP may be usedto deliver to a recipient cell an agent, such as prophylactic agent,therapeutic agent or diagnostic agent, or an enclosed circular or linearDNA or RNA molecule.

In some embodiments, VLPs may have modified immunogenicity and/orantigenicity with respect to the wild type VLPs. The VLPs may, forexample, be assembled from capsomers having a variant capsid proteinwith modified immunogenicity and/or antigenicity. In some embodiments, avariant capsid protein with modified immunogenicity and/or antigenicityis one that is modified naturally or synthetically, such as mutated,substituted, deleted, pegylated or inserted, at an amino acid to reduceor prevent recognition of the capsid protein by pre-existing, such asendogenous, viral serotype-specific antibodies. A variant capsid proteinmay be a human papillomavirus (HPV) L1 variant, a non-humanpapillomavirus L1 variant, or a papillomavirus L1 variant based on acombination of amino acids from different HPV serotypes.

In some embodiments, a VLP is a papilloma virus VLP. The VLP may be ahuman papilloma virus VLP, such as derived from a virus that can infecthuman, while in other embodiments, the VLP may be a non-human papillomavirus VLP. Examples of non-human VLPs include those derived from,without limitation, bovine papilloma viruses, murine papilloma viruses,cotton-rabbit papilloma viruses and macaque or rhesus papilloma virusparticles. In some embodiments, the VLPs are bovine papilloma virusviral-like nanoparticles, such as type 1 viral-like nanoparticles, suchas assembled from BPV L1 capsid proteins or a combination of BPV L1 andBPV L2 capsid proteins.

In some embodiments, a capsid protein refers to a protein monomer,several of which form a capsomer oligomer. In some embodiments, acapsomer refers to the basic oligomeric structural unit of a viralcapsid, which is an outer covering of protein that protects the geneticmaterial of a virus. Capsid proteins may include in some embodiments,papillomavirus L1 major capsid proteins and papillomavirus L2 minorcapsid proteins. In some embodiments, the VLPs contain only L1 capsidproteins, while in other embodiments, the VLPs contain a mixture, orcombination, of L1 and L2 capsid proteins.

In some embodiments, the percentage of L1 capsid proteins in avirus-like particle is greater than the percentage of L2 capsid proteinsin the virus-like particle. For example, in some embodiments, thepercentage of L1 capsid proteins in a virus-like particle is 80% to 100%of the total number of capsid proteins in the virus-like particle. Insome embodiments, the percentage of L1 capsid proteins in a virus-likeparticle is at least or is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100%. In some embodiments, the percentage of L2capsid proteins in a virus-like particle is 1% to 25% of the totalnumber of capsid proteins in the virus-like particle. For example, insome embodiments, the percentage of L2 capsid proteins in a virus-likeparticle is at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

In some embodiments, a virus-like particle contains 12 to 72 L2proteins. In some embodiments, a virus-like particle contains 360 L1proteins and 12 to 72 L2 proteins. In some embodiments, capsid proteinsassemble into viral-like nanoparticles having a diameter of 20 to 60 nm.For example, capsid proteins may assemble into viral-like nanoparticleshaving a diameter of at least or about 20, 25, 30, 35, 40, 45, 50, 55 or60 nm.

In some embodiments, the targeting molecule is not or does not include ananocarrier. In some embodiments, the targeting molecule is not or doesnot include a virus-like particle, a nanoparticle, a liposome, a quantumdot, or a combination thereof.

In some embodiments, the targeting molecule is a DARPin (designedankyrin repeat protein). Typically, DARPins are derived from naturalankyrin repeat proteins and bind to proteins including e.g., humanreceptors, cytokines, kinases, human proteases, viruses and membraneproteins (Molecular Partners AG Zurich Switzerland; see Chapter 5.“Designed Ankyrin Repeat Proteins (DARPins): From Research to Therapy”,Methods in Enzymology, vol 503: 101^(˜)134 (2012); and “EfficientSelection of DARPins with Sub-nanomolar Affinities using SRP PhageDisplay”, J. Mol. Biol. (2008) 382, 1211-1227, the entire disclosures ofwhich are hereby incorporated by reference. In some embodiments, theDARPin is an antibody mimetic protein having high specificity and highbinding affinity to a target protein, which is prepared via geneticengineering. In some embodiments, DARPins have a structure comprising atleast 2 ankyrin repeat motifs, for example, comprising at least 3, 4 or5 ankyrin repeat motifs. The DARPins can have any suitable molecularweight depending on the number of repeat motifs. For example, theDARPins including 3, 4 or 5 ankyrin repeat motifs may have a molecularweight of about 10 kDa, about 14 kDa, or about 18 kDa, respectively.

In some embodiments, the DARPin includes a core part that providesstructure and a target binding portion that resides outside of the coreand binds to a target. In some embodiments, the structural core includesa conserved amino acid sequence and the target binding portion includesan amino acid sequence that differs depending on the target.

In some embodiments, the conjugate contains a number of dye residues pertargeting molecule that is from or from about 1 to about 1000, such asfrom or from about 1 to about 100, from or from about 1 to about 50,from or from about 1 to about 25, from or from about 1 to about 10, fromor from about 1 to about 5. In some embodiments, the ratio of dyemolecules to targeting molecule is or is about 2:1, 3:1, 4:1, 5:1, 10:1,15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1,400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1,900:1, 950:1 or 1000:1, or is between or between about any two of suchvalues. In some embodiments, the targeting molecule may contain up to 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 dyemolecules. In some embodiments, the targeting molecule may contain morethan 1000 dye molecules or less than 10 dye molecules.

In some embodiments, such as when the targeting molecule is apolypeptide, such as an antibody or antigen-binding antibody fragment,the number of dye molecule per targeting molecule can be from or fromabout 2 to about 5, such as from or from about 2 to about 4, for exampleabout 3 or 3. In some embodiments, for example where the targetingmolecule is a nanoparticle, such as a virus-like particle (VLP), thenumber of dye molecules to targeting molecule can be from or from about10 to about 1000, 10 to about 500, 50 to about 500, or 50 to about 1000.Thus, in some embodiments, the targeting molecule may contain about 10to about 1000 dye molecules.

In some embodiments, such as where the targeting molecule is a VLP, morethan one dye molecule may be conjugated to a single capsid protein. Forexample, a single capsid protein, such as LI or L2 capsid protein, maybe conjugated to 1 to 5, such as 1, 2, 3, 4 or 5, dye molecules. Thus,more than one amino acid of a capsid protein may be conjugated to a dyemolecule. In some embodiments, a single capsid protein may be conjugatedto 1 to 2, 1 to 3, or 2 to 3 dye molecules. Thus, a dye molecule may beconjugated to 1, 2, 3, 4 or 5 different amino acids, such as lysine,arginine and/or histidine, or other amino acid, of a single capsidprotein.

3. Additional Dye for Imaging

In some embodiments, the conjugate optionally can include an additionaldye such that the targeting molecule can be conjugated to two or moredifferent fluorescent dyes. For example, provided is a conjugatecontaining a targeting molecule conjugated to a first dye that is aphthalocyanine dye, such as any described above, e.g., IR700, and asecond fluorescent dye that is different than the first dye. In oneaspect, the conjugate is of Formula (II):

[D₁-(L₁)_(n)]_(p)-A-[(L₂)_(m)-D₂]_(o)   (II)

wherein:

-   -   A is a targeting molecule that can bind to cells or tissues;    -   L₁ and L₂ are each an independently selected linker for each o        or p, wherein each L₁ and L₂ are as defined above for L;    -   n and m are independently 1 or 2;    -   D₁ is an independently selected hydrophilic phthalocyanine dye        for each p, wherein D₁ is as defined above for D;    -   D₂ is an independently selected fluorescent dye for each o; and    -   p and o are independently 1, 2, 3, 4, 5 or greater than 5, such        as up to 1000. For example, p    -   and o can each independently be 1 to 1000, such as generally 1        to 10 or 2 to 5.

In some embodiments, the first dye D₁ is a phthalocyanine dye, such as anear infrared (NIR) phthalocyanine dye, such as any of the dyesdescribed above. In some embodiments, the phthalocyanine dye is orcomprises a photosensitizer compound such that it is capable ofexhibiting phototoxic activity upon irradiation with near-infraredlight. In some embodiments, D₁ is IR700.

In some embodiments, the second dye D₂ is selected to offer betterfluorescence for visualization than the first dye D₁ (e.g., IR700).Thus, in some aspects, the compound of formula II is used for bothfluorescence imaging and photoimmunotherapy. For example, irradiatingthe lesion or tumor emits a fluorescence signal from the secondfluorescent dye to effect detection of the presence of the conjugate atthe lesion or tumor in the subject. In some embodiments, the conjugatecan be used to both monitor the binding of the dye to the target site(e.g., tumor) with fluorescence imaging of D₂ and to eradicate cellsassociated with a disease or condition, e.g., cells of a tumor, usingphotoimmunotherapy by activation of D₁ (e.g., IR700). In someembodiments, the second dye D₂ exhibits one or more spectral propertiesselected from among fluorescent quantum yield (e.g., in water),extinction coefficient, Stokes shift, absorption and emission at longwavelength, and photostability that is greater compared to thecorresponding spectral property of D₁. In some embodiments, D₂ is notIR700.

In some embodiments, the additional dye D₂ is a fluorescent dye. In someembodiments, D₂ can be, but is not limited to, hydroxycoumarin, CascadeBlue, Dylight 405 Pacific Orange, Alexa Fluor 430, Fluorescein, OregonGreen, Alexa Fluor 488, BODIPY 493, 2,7-Diochlorofluorescien, ATTO 488,Chromeo 488, Dylight 488, HiLyte 488, Alexa Fluor 532, Alexa Fluor 555,ATTO 550, BODIPY TMR-X, CF 555, Chromeo 546, Cy3, TMR, TRITC, Dy547,Dy548, Dy549, HiLyte 555, Dylight 550, BODIPY 564, Alexa Fluor 568,Alexa Fluor 594, Rhodamine, Texas Red, Alexa Fluor 610, Alexa Fluor 633,Dylight 633, Alexa Fluor 647, APC, ATTO 655, CF633, CF640R, Chromeo642,Cy5, Dylight 650, Alexa Fluor 680, IRDye 680, Alexa Fluor 700, Cy5.5,ICG, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7, Cy7.5, Alexa Fluor790, Dylight 800, IRDye 800, Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655,Qdot® 705, or Qdot® 800.

In some embodiments, D₂ absorbs light and emits fluorescence at longerwavelengths where tissue autofluorescence is low, essentially absent oreliminated or is absent or eliminated. Thus, in some aspects, D₂facilitates a deep tissue penetration of imaging. In some embodiments,D₂ is a near-infrared (NIR) dye that has an absorption and emissionwavelength in the NIR spectrum between or between about 650 and 1450 nm,such as between or between about 650 and 900 nm, between or betweenabout 700 and 1000 nm, or between or between about 750 and 950 nm. Insome embodiments, D₂ is a second near-infrared (NIR-II) dye that has anabsorption and emission wavelength in the NIR-II spectrum between orbetween about 1000 and 1700 nm, such as between or between about 1000and 1400 nm. In some embodiments, D₂ is a dye that has an absorption andemission wavelength in the visible spectrum between or between about 300and 750 nm, such as between or between about 400 and 600 nm or betweenor between about 400 and 700 nm. In some embodiments, the targetingmolecule conjugate contains two dyes with different emission andexcitation wavelengths. In some embodiments, the additional dye has longwavelength excitation and emission properties. In some embodiments, anyof the provided methods can further comprise imaging the lesion or tumorin the subject by irradiating or illuminating the tumor at a wavelengthcapable of being absorbed by the second dye.

In some embodiments, the additional dye has a large extinctioncoefficient at the excitation wavelength. In some embodiments, D₂ has aextinction coefficient in water of above about 10,000 Mol⁻¹cm⁻¹, such asabove about 25,000 Mol⁻¹cm⁻¹, above about 50,000 Mol⁻¹cm⁻¹, above about75,000 Mol⁻¹cm⁻¹, above about 100,000 Mol⁻¹cm⁻¹, above about 150,000Mol⁻¹cm⁻¹, above about 200,000 Mol⁻¹cm⁻¹, above about 250,000 Mol⁻¹cm⁻¹,or above about 300,000 Mol⁻¹cm⁻¹.

In some embodiments, the second dye D₂ has a higher fluorescent quantumyield when conjugated to proteins than does the first dye D₁. In someembodiments, the additional dye has a high fluorescent quantum yield inwater. In some embodiments, the second dye D₂ has a quantum yield inwater that is greater than 5%, such as great than 10%, greater than 15%,greater than 20% or greater than 25%, greater than 30%, greater than40%, or greater than 50% or greater.

In some embodiments, the second dye D₂ has a large Stokes shift(difference between EX_(max) and EM_(max)). In some embodiments, theadditional dye D₂ exhibits a Stokes shift that is greater than 15 nm, 20nm, 25 nm, such as great than 30 nm, greater than 40 nm, greater than 50nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greaterthan 90 nm or greater than 100 nm.

In some embodiments, the additional dye D₂ can be ICG, IRDye 680, AlexaFluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800,or IRDye 800. In some embodiments, the additional dye D₂ can be ICG,IRDye 680, Alexa Fluor 750, Dylight 755, IRDye 750, Cy7.5, Alexa Fluor790, Dylight 800, or IRDye 800. In some embodiments, the additional dyeD₂ can be Alexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

In some embodiments, the conjugate contains a number of second dye, D₂,residues per targeting molecule that is from or from about 1 to about1000, such as from or from about 1 to about 100, from or from about 1 toabout 50, from or from about 1 to about 25, from or from about 1 toabout 10, or from or from about 1 to about 5. In some embodiments, theratio of second dye molecules to targeting molecule is or is about 2:1,3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1,250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1,750:1, 800:1, 850:1, 900:1, 950:1 or 1000:1, or is between or betweenabout any two of such values. In some embodiments, the ratio of seconddye molecules to targeting molecule is 1 to 10 or 1 to 5 second dyemolecules per targeting molecule.

II. Pharmaceutical Compositions and Articles of Manufacture

Provided herein are pharmaceutical compositions containing aphthalocyanine-dye targeting molecule conjugate (e.g., IR700-antibodyconjugate). In some embodiments, the compositions can be used in methodsof PIT as described herein. The phthalocyanine dye-targeting moleculeconjugate, for example, IR700-antibody conjugate. In some embodiments,the compositions can be provided in combination with another therapeuticagent (e.g., an immune modulating agent or anti-cancer agent). In someembodiments, the phthalocyanine dye-targeting molecule conjugate andother therapeutic agent, such as one or both of an immune modulatingagent or anti-cancer agent, can be packaged as an article of manufactureas separate compositions for administration together, sequentially orintermittently. The combinations can be packaged as a kit.

1. Compositions, Formulations and Dosage Forms

In some embodiments, the compounds, such as conjugate, can be formulatedin a pharmaceutically acceptable buffer, such as that containing apharmaceutically acceptable carrier or vehicle. Generally, thepharmaceutically acceptable carriers or vehicles, such as those presentin the pharmaceutically acceptable buffer, are can be any known in theart. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition (1995), describes compositionsand formulations suitable for pharmaceutical delivery of one or moretherapeutic compounds. Pharmaceutically acceptable compositionsgenerally are prepared in view of approvals for a regulatory agency orother agency prepared in accordance with generally recognizedpharmacopeia for use in animals and in humans.

Pharmaceutical compositions can include carriers such as a diluent,adjuvant, excipient, or vehicle with which the compound is administered.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the compound,generally in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is atypical carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Compositions can contain along with an activeingredient: a diluent such as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acacia, gelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, andethanol. A composition, if desired, also can contain minor amounts ofwetting or emulsifying agents, or pH buffering agents, for example,acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

In some embodiments, pharmaceutical preparation can be in liquid form,for example, solutions, syrups or suspensions. Such liquid preparationscan be prepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). In some cases,pharmaceutical preparations can be presented in lyophilized form forreconstitution with water or other suitable vehicle before use.

In some embodiments, the nature of the pharmaceutically acceptablebuffer, or carrier, depends on the particular mode of administrationbeing employed. For instance, in some embodiments, parenteralformulations may comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose, orglycerol as a vehicle. In some embodiments, for solid compositions, forexample powder, pill, tablet, or capsule forms, non-toxic solid carrierscan include, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can in someembodiments contain minor amounts of non-toxic auxiliary substances,such as wetting or emulsifying agents, preservatives, and pH bufferingagents, for example sodium acetate or sorbitan monolaurate.

The compounds can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administrate, as well as transdermal patch preparationand dry powder inhalers. Typically, the compounds are formulated intopharmaceutical compositions using techniques and procedures well knownin the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms,Fourth Edition, 1985, 126). Generally, the mode of formulation is afunction of the route of administration.

Compositions can be formulated for administration by any route known tothose of skill in the art including intramuscular, intravenous,intradermal, intralesional, intraperitoneal injection, subcutaneous,intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local,otic, inhalational, buccal (e.g., sublingual), and transdermaladministration or any route. Other modes of administration also arecontemplated. Administration can be local, topical or systemic dependingupon the locus of treatment. Local administration to an area in need oftreatment can be achieved by, for example, but not limited to, localinfusion during surgery, topical application, e.g., in conjunction witha wound dressing after surgery, by injection, by means of a catheter, bymeans of a suppository, or by means of an implant.

Parenteral administration, generally characterized by injection, eithersubcutaneously, intramuscularly, intratumorally, intravenously orintradermally is contemplated herein. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain anactivator in the form of a solvent such as pH buffering agents, metalion salts, or other such buffers. The pharmaceutical compositions alsomay contain other minor amounts of non-toxic auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, stabilizers,solubility enhancers, and other such agents, such as for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) also is contemplated herein. The percentage of activecompound contained in such parenteral compositions is highly dependenton the specific nature thereof, as well as the activity of the compoundand the needs of the subject.

Injectables are designed for local and systemic administration.Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products, such as lyophilizedpowders, ready to be combined with a solvent just prior to use,including hypodermic tablets, sterile suspensions ready for injection,sterile dry insoluble products ready to be combined with a vehicle justprior to use and sterile emulsions. The solutions may be either aqueousor nonaqueous. If administered intravenously, suitable carriers includephysiological saline or phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents, such as glucose,polyethylene glycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers, which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

The composition can be formulated for single dosage administration orfor multiple dosage administration. The agents can be formulated fordirect administration. The composition can be provided as a liquid orlyophilized formulation. Where the composition is provided inlyophilized form it can be reconstituted just prior to use by anappropriate buffer, for example, a sterile saline solution.

Compositions also can be administered with other biologically activeagents, either sequentially, intermittently or in the same composition.Administration also can include controlled release systems includingcontrolled release formulations and device controlled release, such asby means of a pump.

The most suitable route in any given case depends on a variety offactors, such as the nature of the disease, the progress of the disease,the severity of the disease and the particular composition which isused. For example, compositions are administered sytemically, forexample, via intravenous administration. Subcutaneous methods also canbe employed, although increased absorption times can be necessary toensure equivalent bioavailability compared to intravenous methods.

Pharmaceutical compositions can be formulated in dosage formsappropriate for each route of administration. Pharmaceutically andtherapeutically active compounds and derivatives thereof are typicallyformulated and administered in unit dosage forms or multiple dosageforms. Each unit dose contains a predetermined quantity oftherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Unit dosage forms, include, but are notlimited to, tablets, capsules, pills, powders, granules, sterileparenteral solutions or suspensions, and oral solutions or suspensions,and oil water emulsions containing suitable quantities of the compoundsor pharmaceutically acceptable derivatives thereof. Unit dose forms canbe contained ampoules and syringes or individually packaged tablets orcapsules. Unit dose forms can be administered in fractions or multiplesthereof. A multiple dose form is a plurality of identical unit dosageforms packaged in a single container to be administered in segregatedunit dose form. Examples of multiple dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit doses that are not segregated inpackaging. Generally, dosage forms or compositions containing activeingredient in the range of 0.005% to 100% with the balance made up fromnon-toxic carrier can be prepared. Pharmaceutical compositions can beformulated in dosage forms appropriate for each route of administration.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art. The unit-doseparenteral preparations are packaged in an ampoule, a vial or a syringewith a needle. The volume of liquid solution or reconstituted powderpreparation, containing the pharmaceutically active compound, is afunction of the disease to be treated and the particular article ofmanufacture chosen for package. All preparations for parenteraladministration must be sterile, as is known and practiced in the art. Insome embodiments, the compositions can be provided as a lyophilizedpowder, which can be reconstituted for administration as solutions,emulsions and other mixtures. They may also be reconstituted andformulated as solids or gels. The lyophilized powders can be preparedfrom any of the solutions described above.

The sterile, lyophilized powder can be prepared by dissolving aphthalocyanine dye-targeting molecule conjugate in a buffer solution.The buffer solution may contain an excipient which improves thestability of other pharmacological components of the powder orreconstituted solution, prepared from the powder.

In some embodiments, subsequent sterile filtration of the solutionfollowed by lyophilization under standard conditions known to those ofskill in the art provides the desired formulation. Briefly, thelyophilized powder is prepared by dissolving an excipient, such asdextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose,sucrose or other suitable agent, in a suitable buffer, such as citrate,sodium or potassium phosphate or other such buffer known to those ofskill in the art. Then, a selected enzyme is added to the resultingmixture, and stirred until it dissolves. The resulting mixture issterile filtered or treated to remove particulates and to ensuresterility, and apportioned into vials for lyophilization. Each vial cancontain a single dosage (1 mg-1 g, generally 1-100 mg, such as 1-5 mg)or multiple dosages of the compound. The lyophilized powder can bestored under appropriate conditions, such as at about 4° C. to roomtemperature. Reconstitution of this lyophilized powder with a buffersolution provides a formulation for use in parenteral administration.The precise amount depends upon the indication treated and selectedcompound. Such amount can be empirically determined.

In some embodiments, the pH of the composition is between or betweenabout 6 and 10, such as between or between about 6 and 8, between orbetween about 6.9 and 7.3, such as about pH 7.1. In some embodiments,the pH of the pharmaceutically acceptable buffer is at least or about 5,at least or about 6, at least or about 7, at least or about 8, at leastor about 9 or at least or about 10, or is 7.1.

The compositions can be formulated for single dosage administration orfor multiple dosage administration. The agents can be formulated fordirect administration.

In some embodiments, the compositions provided herein are formulated inan amount for direct administration of the active compound, such asphthalocyanine dye-targeting molecule conjugate, in a range from or fromabout 0.01 mg to about 3000 mg, from about 0.01 mg to about 1000 mg,from about 0.01 mg to about 500 mg, from about 0.01 mg to about 100 mg,from about 0.01 mg to about 50 mg, from about 0.01 mg to about 10 mg,from about 0.01 mg to about 1 mg, from about 0.01 mg to about 0.1 mg,from about 0.1 mg to about 2000 mg, from about 0.1 mg to about 1000 mg,from about 0.1 mg to about 500 mg, from about 0.1 mg to about 100 mg,from about 0.1 mg to about 50 mg, from about 0.1 mg to about 10 mg, fromabout 0.1 mg to about 1 mg, from about 1 mg to about 2000 mg, from about1 mg to about 1000 mg, from about 1 mg to about 500 mg, from about 1 mgto about 100 mg, from about 1 mg to about 10 mg, from about 10 mg toabout 2000 mg, from about 10 mg to about 1000 mg, from about 10 mg toabout 500 mg, from about 10 mg to about 100 mg, from about 100 mg toabout 2000 mg, from about 100 mg to about 1000 mg, from about 100 mg toabout 500 mg, from about 500 mg to about 2000 mg, from about 500 mg toabout 1000 mg, and from about 1000 mg to about 3000 mg. In someembodiments, the volume of the composition can be 0.5 mL to 1000 mL,such as 0.5 mL to 100 mL, 0.5 mL to 10 mL, 1 mL to 500 mL, 1 mL to 10mL, such as at least or about at least or about or 0.5 mL, 1 mL, 2 mL, 3mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40mL, 50 mL or more. For example, the composition is formulated for singledosage administration of an amount between or between about 100 mg and500 mg, or between or between about 200 mg and 400 mg. In someembodiments, the composition is formulated for single dosageadministration of an amount between or between about 500 mg and 1500 mg,800 mg and 1200 mg or 1000 mg and 1500 mg. In some embodiments, thevolume of the composition is between or between about 10 mL and 1000 mLor 50 mL and 500 mL; or the volume of the composition is at least 10 mL,20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300mL, 400 mL, 500 mL or 1000 mL.

In some embodiments, the entire vial contents of the formulations can bewithdrawn for administration, or can be divided up into a plurality ofdosages for multiple administrations. Upon withdrawal of an amount ofdrug for administration, the formulation can be further diluted ifdesired, such as diluted in water, saline (e.g., 0.9%) or otherphysiological solution.

In some embodiments, also provided are compositions containing an immunemodulating agent or anti-cancer agent, which can be prepared in accordwith known or standard formulation guidelines, such as described above.In some embodiments, the immune modulating agent, anti-cancer agentand/or phthalocyanine dye-targeting molecule conjugate (e.g.,IR700-targeting molecule, such as IR700-antibody conjugate) areformulated as separate compositions. In some embodiments, the immunemodulating agent is provided as a separate composition from thephthalocyanine dye-targeting molecule conjugate, and the twocompositions are administered separately. In some embodiments, theanti-cancer agent is provided as a separate composition from thephthalocyanine dye-targeting molecule conjugate, and the twocompositions are administered separately. The compositions can beformulated for parenteral delivery (i.e. for systemic delivery). Forexample, the compositions or combination of compositions are formulatedfor subcutaneous delivery or for intravenous delivery. The agents, suchas a phthalocyanine dye-targeting molecule conjugate, an immunemodulating agent, and/or an anti-cancer agent can be administered bydifferent routes of administration.

3 Packaging and Articles of Manufacture

Also provided are articles of manufacture containing packagingmaterials, any pharmaceutical compositions or combinations providedherein, and a label that indicates that the compositions andcombinations are to be used for treatment of cancers. Exemplary articlesof manufacture are containers including single chamber and dual chambercontainers. The containers include, but are not limited to, tubes,bottles and syringes. The containers can further include a needle forsubcutaneous administration.

In some embodiments, the agents can be provided separately for packagingas articles of manufacture. In some embodiments, the article ofmanufacture contains pharmaceutical compositions containing thephthalocyanine dye-targeting molecule conjugate, such as aIR700-antibody conjugate, and the immune modulatory agent. In someembodiments, the article of manufacture contains pharmaceuticalcompositions containing the phthalocyanine dye-targeting moleculeconjugate (e.g., IR700-antibody conjugate) and an anti-cancer agent. Insome embodiments, the article of manufacture contains pharmaceuticalcompositions containing the phthalocyanine dye-targeting moleculeconjugate (e.g., IR700-antibody conjugate), the immune modulatory agent,and a further anti-cancer agent.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, for example, U.S. Pat.Nos. 5,323,907, 5,052,558 and 5,033,252, each of which is incorporatedherein in its entirety. Examples of pharmaceutical packaging materialsinclude, but are not limited to, blister packs, bottles, tubes,inhalers, pumps, bags, vials, containers, syringes, bottles, and anypackaging material suitable for a selected formulation and intended modeof administration and treatment. The choice of package depends on theagents. In general, the packaging is non-reactive with the compositionscontained therein.

The components can be packaged in the same of different container. Forexample, in some embodiments, the components are separately packaged inthe same container. Generally, examples of such containers include thosethat have an enclosed, defined space that contains the phthalocyaninedye-targeting molecule conjugate, and a separate enclosed, defined spacecontaining the other components or component such that the subsequentareas are separated to permit the components to be separatelyadministered. Any container or other article of manufacture iscontemplated, so long as the agents are separated from the othercomponents prior to administration. For suitable embodiments see e.g.,containers described in U.S. Pat. Nos. 3,539,794 and 5,171,081. In someembodiments, a plurality of containers are provided, each separatelycontaining a phthalocyanine dye-targeting molecule conjugate, an immunemodulating agent or an anti-cancer agent. In such examples, theplurality of containers can be packaged together as a kit.

In some embodiments, a container containing the phthalocyaninedye-targeting molecule conjugate is contained in a light-protectedcontainer. In some embodiments, the container is a vial, such as adepyrogenated, glass vial. In some embodiments, the container, such as avial, blocks light of a particular wavelength, such as a wavelength oflight that is absorbed by the dye or dye-targeting molecule conjugate.In some embodiments, the conjugate is protected from light usingcontainers that protect contents from light, or certain wavelengths orintensities of light. For example, in some embodiments, the containerhas a light transmittance of no more than 50%, no more than 40%, no morethan 30%, no more than 20%, no more than 10%, no more than 5%, or nomore than 1%. In some embodiments, the container protects fromtransmittance of light having a wavelength between or between about 500nm and 725 nm, such as between or between about 650 nm and 725 nm, ordoes not transmit an intensity of light greater than 700 lux, 600 lux,500 lux, 400 lux, 300 lux, 200 lux, or 100 lux. In some embodiments, thecontainer is green, amber, translucent, opaque, or is wrapped in anopaque material, such as a foil, such as aluminum foil. In someembodiments, the container is sterile or depyrogenated.

In some embodiments, the conjugates are provided in a plurality ofsealable containers. For example, the containers can each individuallycomprising a fraction of a single administration dose of a compositioncontaining a conjugate that includes a phthalocyanine dye linked to atargeting molecule. In some embodiments, the combined amount of theconjugate in the plurality of sealable containers is between or betweenabout 100 mg and 1500 mg, or 100 mg and 1200 mg. In some embodiments,the combined amount of the conjugate in the plurality of sealablecontainer is between or between about 100 mg and 500 mg, between orbetween about 200 mg and 400 mg, between or between about 500 mg and1500 mg, between or between about 800 mg and 1200 mg or between orbetween about 1000 mg and 1500 mg.

In some embodiments, the article of manufacture contains packagingmaterial and a label or package insert containing instructions forcombining the contents of the plurality of vials to prepare a singledosage formulation of the composition.

In some embodiments, the containers are further packaged to protect thecontents from light. In some embodiments, a packaging system is providedthat includes an internal packaging material comprising a containercomprising the phthalocyanine dye-targeting molecule conjugate (e.g.,IR700-targeting molecule conjugate, such as IR700-antibody conjugate),and optionally a container containing an immune modulating agent oranti-cancer agent. In some embodiments, the internal packaging materialhas a light transmittance of less than 20%, such as less than 15%, lessthan 10%, less than 5%, or less than 1%. In some embodiments, thepackaging system includes an external packaging material comprising theinternal packaging material. In some embodiments, the external packagingmaterial has a light transmittance of less than 20%, such as less than15%, less than 10%, less than 5%, or less than 1%. In some embodiments,the internal or external packaging material includes an opaque foil,such as aluminum foil. In some embodiments, the secondary packagingmaterial is an aluminum pouch. In some embodiments, the externalpackaging material comprises cardboard.

In some embodiments, the internal and/or external packaging material issuitable for storage of the conjugate. In some embodiments, the internaland/or external packaging material is suitable for shipping of theconjugate.

Selected compositions including articles of manufacture thereof also canbe provided as kits. Kits can include a pharmaceutical compositiondescribed herein and an item for administration provided as an articleof manufacture. The kit can, optionally, include instructions forapplication including dosages, dosing regimens and instructions formodes of administration. Kits also can include a pharmaceuticalcomposition described herein and an item for diagnosis.

In some embodiments, the compositions used for administration of agents,such as the phthalocyanine dye-targeting molecule conjugate contain aneffective amount of each agent along with conventional pharmaceuticalcarriers and excipients appropriate for the type of administrationcontemplated.

In some embodiments, a single dosage amount of the agent, such as thephthalocyanine dye-targeting molecule conjugate, is comprised within asingle container, such as a container in which the agent is stored. Insome embodiments, a single dosage amount of the agent is comprised in aplurality of containers. Thus, in some embodiments, a plurality ofcontainers, such as vials, are combined, in a container to be used foradministration of the agent, such as an intravenous (IV) bag. In someembodiments, the container used for administration, such as IV bag, isprepared by opening one or a plurality of containers comprising theagent and placing the contents in the bag, such as until a desired doseof the agent for administration, e.g., infusion, is achieved. During thepreparation of the administration container, such as IV bag, lightprecautions were taken to avoid exposure of the agent to light, such asthe various light precautions described herein.

III. Methods of Treatment

In some embodiments, provided are methods for using and uses of thecompositions containing a phthalocyanine-dye targeting moleculeconjugate (e.g., IR700-targeting molecule conjugate, such asIR700-antibody conjugate) for PIT. In some embodiments, thephthalocyanine-dye targeting molecule conjugate targets to a cell orpathogen associated with a disease or condition, such as via binding toa cell surface protein or cell surface receptor expressed on a cell.Such methods and uses include therapeutic methods and uses, for example,involving administration of the molecules to a subject having a disease,condition or disorder followed by irradiation to achievephotoimmunotherapy (PIT), thereby resulting in photolysis of such cellsor pathogens to effect treatment of the disease or disorder. In someembodiments, the methods can be used for treating a tumor or a cancer,whereby an administered phthalocyanine-dye targeting molecule conjugate(IR700-targeting molecule conjugate, such as IR700-antibody conjugate)is targeted to a cell associated with a tumor, thereby resulting inphotolysis of such cell and, in some cases, resulting in treatment ofthe tumor. Uses include uses of the compositions in such methods andtreatments, and uses of such compositions in the preparation of amedicament in order to carry out such therapeutic methods. In someembodiments, the methods and uses thereby treat the disease or conditionor disorder, such as a tumor or cancer, in the subject.

In some embodiments, the methods include administration of aphthalocyanine dye-targeting molecule conjugate (e.g., IR-700 antibodyconjugate) to the subject under conditions in which, generally, a celltargeted for killing is contacted with the conjugate. In someembodiments, the methods result in the binding of the targeting molecule(e.g., antibody) portion of the conjugate to a cell surface proteinassociated with a tumor or cancer. After contacting or administering theconjugate, a local area of the subject containing the targeted cells,e.g., a cell or cells associated with a tumor, is exposed or irradiatedwith light absorbed by the dye, generally NIR light, thereby activatingthe conjugate to effect specific cell killing. In some embodiments,irradiation is performed at a wavelength of 600 nm to 850 nm at a doseof at least 1 J cm⁻² or at least 1 J/cm of fiber length. In someembodiments, the methods of administering a phthalocyanine dye-targetingmolecule conjugate (e.g., IR-700 antibody conjugate) include methodsdescribed in U.S. Pat. No. 8,524,239 or U.S. publication No.US2014/0120119.

A. Tumors and Subjects to be Treated

In some embodiments, the lesion is a tumor. In some embodiments, thetumor is a cancer. In some embodiments, the cancer is a cancer of thehead and neck, breast, liver, colon, ovary, prostate, pancreas, brain,cervix, bone, skin, lung, or blood. In some embodiments, cancer mayinclude a malignant tumor characterized by abnormal or uncontrolled cellgrowth. Other features that may be associated with cancer includemetastasis, interference with the normal functioning of neighboringcells, release of cytokines or other secretory products at abnormallevels and suppression or aggravation of inflammatory or immunologicalresponse, invasion of surrounding or distant tissues or organs, such aslymph nodes, etc. Metastatic disease may refer to cancer cells that haveleft the original tumor site and migrated to other parts of the body,for example via the bloodstream or lymph system. In some embodiments, acell targeted by the disclosed methods is a cancer cell or an immunecell. In some embodiments, the cancer cell is a cancer stem cell. Insome embodiments, a cell targeted by the disclosed methods is a cellthat is a cancer cell, a tumor cell, an inflammatory cell, an immunecell, a neuron, a stem cell, a proliferating cell, or a cell in ahyperplasia.

The target cell can be a cell that is not desired or whose growth is notdesired, such as a tumor or cancer cell. In some embodiments, the cellscan be growing in culture, or present in a mammal to be treated, such asa subject with cancer. Any target cell can be treated with the claimedmethods. In some embodiments, the target cell expresses a cell surfaceprotein that is not substantially found on the surface of other normalcells. In some embodiments, an antibody can be selected thatspecifically binds to such protein, and a phthalocyanine dye-antibodyconjugate may be generated for that protein. In some embodiments, thecell surface protein is a tumor-specific protein. In some embodiments,the cell surface protein is CD25, which can be used to target cellsassociated with undesired transplant rejection.

In some embodiments, the tumor cell is a cancer cell, such as a cell ina subject with cancer. Exemplary cells that can be targeted in thedisclosed methods include cells of the following tumors: a liquid tumorsuch as a leukemia, including acute leukemia (such as acute lymphocyticleukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia and chronic lymphocyticleukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin'slymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease). In some embodiments, the cell is a solid tumor cell, such as asarcoma or carcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, hepatocellular carcinoma, lung cancer, colorectal cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, forexample adenocarcinoma of the pancreas, colon, ovary, lung, breast,stomach, prostate, cervix, or esophagus, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer, testicular tumor, bladder carcinoma, CNS tumors, suchas a glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma. In some embodiments, the cancer is a squamous cellcarcinoma of the head and neck.

Exemplary tumors, such as cancers, that can be treated with the claimedmethods include solid tumors, such as breast carcinomas, such as lobularand duct carcinomas, sarcomas, carcinomas of the lung, such as non-smallcell carcinoma, large cell carcinoma, squamous carcinoma, andadenocarcinoma, mesothelioma of the lung, colorectal adenocarcinoma,stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma, such asserous cystadenocarcinoma and mucinous cystadenocarcinoma, ovarian germcell tumors, testicular carcinomas and germ cell tumors, pancreaticadenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma,bladder carcinoma, including, for instance, transitional cell carcinoma,adenocarcinoma, and squamous carcinoma, renal cell adenocarcinoma,endometrial carcinomas, including, for instance, adenocarcinomas andmixed Mullerian tumors (carcinosarcomas), carcinomas of the endocervix,ectocervix, and vagina, such as adenocarcinoma and squamous carcinoma ofeach of same, tumors of the skin, such as squamous cell carcinoma, basalcell carcinoma, malignant melanoma, skin appendage tumors, Kaposisarcoma, cutaneous lymphoma, skin adnexal tumors and various types ofsarcomas and Merkel cell carcinoma, esophageal carcinoma, carcinomas ofthe nasopharynx and oropharynx, including squamous carcinoma andadenocarcinomas of same, salivary gland carcinomas, brain and centralnervous system tumors, including, for example, tumors of glial,neuronal, and meningeal origin, tumors of peripheral nerve, soft tissuesarcomas and sarcomas of bone and cartilage, and lymphatic tumors,including B-cell and T-cell malignant lymphoma. In some embodiments, thetumor is an adenocarcinoma.

The methods can also be used to treat liquid tumors, such as alymphatic, white blood cell, or other type of leukemia. In someembodiments, the tumor treated is a tumor of the blood, such as aleukemia, for example acute lymphoblastic leukemia (ALL), chroniclymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CIVIL), hairy cell leukemia (HCL), T-cellprolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia,and adult T-cell leukemia, lymphomas, such as Hodgkin's lymphoma andnon-Hodgkin's lymphoma, and myelomas.

In some embodiments, the conjugate is targeted to a protein expressed onthe surface of a lesion or on the surface of a cell present in themicroenvironment of the lesion. For example, in some embodiments, theconjugate is targeted to a protein expressed on the surface of a cell inthe tumor or on the surface of a cell in the microenvironment of thetumor. Exemplary of such cell surface proteins are any as describedherein, including those described above.

In some embodiments, the protein on the cell surface of the target cellto be targeted is not present in significant amounts on other cells. Forexample, the cell surface protein can be a receptor that is only foundon the target cell type.

In some embodiments, the protein expressed in the tumor, e.g.,tumor-specific protein, can be HER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2Rareceptor), CD33, CD52, CD133, CD206, CEA, cancer antigen 125 (CA125),alpha-fetoprotein (AFP), Lewis Y, TAG72, vascular endothelial growthfactor (VEGF), CD30, EpCAM, EphA2, Glypican-3, gpA33, mucins, CAIX,PSMA, folate-binding protein, gangliosides (such as GD2, GD3, GM1 andGM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex,CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen, IgE, MUC-1,nuC242, PEM antigen, SK-1 antigen or PD-L1. In some embodiments, thetumor-specific protein is PD-L1, HER1/EGFR, HER2, CD20, CD25, CD33,CD52, prostate specific membrane antigen (PSMA), EpCAM, EphA2, CD206,CD44, CD133, Mesothelin, Glypican-3, or carcinoembryonic antigen (CEA).Other cell surface proteins include any as described above.

In some embodiments, the cell surface protein is associated with atumor, such as is a tumor-specific protein or tumor-specific antigen,such as members of the EGF receptor family (e.g., HER1, 2, 3, and 4) andcytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). In someembodiments, tumor specific proteins are those proteins that are uniqueto cancer cells or are much more abundant on them, as compared to othercells, such as normal cells. For example, HER2 is generally found inbreast cancers, while HER1 is typically found in adenocarcinomas, whichcan be found in many organs, such as the pancreas, breast, prostate andcolon.

Exemplary proteins associated with a tumor that can be found on a targetcell, and to which targeting molecule, e.g. antibody or antibodyfragment, specific for that protein can be used to formulate aphthalocyanine dye-antibody conjugate, include but are not limited to:any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE1, MAGE 2, MAGE 3, and MAGE 4, any of the various tyrosinases, mutantras, mutant p53, p97 melanoma antigen, human milk fat globule (HMFG)which may be associated with breast tumors, any of the various BAGEs(Human B melanoma-Associated Antigen E), including BAGE1 and BAGE2, anyof the various GAGEs (G antigen), including GAGE1, GAGE2-6, variousgangliosides, and CD25.

Other proteins associated with a tumor include the HPV 16/18 and E6/E7antigens associated with cervical cancers, mucin (MUC 1)-KLH antigenwhich may be associated with breast carcinoma, CEA (carcinoembryonicantigen) which may be associated with colorectal cancer, gp100 which maybe associated with for example melanoma, MARTI antigens which may beassociated with melanoma, cancer antigen 125 (CA125, also known as mucin16 or MUC16) which may be associated with ovarian and other cancers,alpha-fetoprotein (AFP) which may be associated with liver cancer, LewisY antigen which may be associated with colorectal, biliary, breast,small-cell lung, and other cancers, tumor-associated glycoprotein 72(TAG72) which may be associated with adenocarcinomas, and the PSAantigen which may be associated with prostate cancer.

Other exemplary proteins associated with a tumor further include, butare not limited to, PMSA (prostate membrane specific antigen), which maybe associated with solid tumor neovasculature, as well prostate cancer,HER-2 (human epidermal growth factor receptor 2) which may be associatedwith breast cancer, ovarian cancer, stomach cancer and uterine cancer,HER-1 which may be associated with lung cancer, anal cancer, andglioblastoma as well as adenocarcinomas, NY-ESO-1 which may beassociated with melanoma, sarcomas, testicular carcinomas, and othercancers, hTERT (aka telomerase), proteinase 3, and Wilms tumor 1 (WT-1).

In some embodiments, the protein associated with a tumor is CD52 and maybe associated with chronic lymphocytic leukemia, CD33 and may beassociated with acute myelogenous leukemia, or CD20 and may beassociated with Non-Hodgkin lymphoma.

Thus, the disclosed methods can be used to treat any cancer thatexpresses a tumor-specific protein. In some embodiments, the tumortherapeutic is an antibody, an antigen binding fragment, a protein, aglycoprotein, a peptide, a polypeptide, a virus, a viral capsid, or aviral particle. In some embodiments, the tumor therapeutic is anantibody or an antigen binding fragment.

In some embodiments, the subject is a human or non-human mammal. In someembodiments, the subject is a human or veterinary subject, such as amouse. In some embodiments, the subject is a mammal, such as a human,who has cancer, or is being treated for cancer. In some embodiments thedisclosed methods are used to treat a subject who has a tumor, such as atumor described herein. In some embodiments, the tumor has beenpreviously treated, such as surgically or chemically removed, and thedisclosed methods are used subsequently to kill any remaining undesiredtumor cells that may remain in the subject.

The disclosed methods can be used to treat any mammalian subject, suchas a human, who has a tumor, such as a cancer, or has had suchpreviously removed or treated. Subjects in need of the disclosedtherapies can include human subjects having cancer, wherein the cancercells express a tumor-specific protein on their surface that canspecifically bind to phthalocyanine dye-targeting molecule conjugate.For example, the disclosed methods can be used as initial treatment forcancer either alone, or in combination with radiation or otherchemotherapy. The disclosed methods can also be used in patients whohave failed previous radiation or chemotherapy. Thus, in someembodiments, the subject is one who has received other therapies, butthose other therapies have not provided a desired therapeutic response.The disclosed methods can also be used in patients with localized and/ormetastatic cancer.

In some embodiments, the method includes selecting a subject that willbenefit from the disclosed therapies, such as selecting a subject havinga tumor that expresses a cell surface protein, such as a tumor-specificprotein, that can specifically bind to a phthalocyanine dye-targetingmolecule conjugate. For example, if the subject is determined to have abreast cancer that expresses HER1, the subject may be selected to betreated with an anti-HER1-IR700 molecule, such as cetuximab-IR700.

B. Dosage and Administration

The compositions provided herein containing a phthalocyaninedye-targeting molecule conjugate (e.g., IR700-antibody conjugate) areadministered in amounts that are sufficient to exert a therapeuticallyuseful effect. Typically, the active agents are administered in anamount that does not result in undesirable side effects of the patientbeing treated, or that minimizes or reduces the observed side effects ascompared to dosages and amounts required for single treatment with oneof the above agents.

Methods of determining optimal dosages of a phthalocyanine dye-targetingmolecule conjugate (e.g., IR700-antibody conjugate) to a patient in needthereof, either alone or in combination with one or more other agents,may be determined by standard dose-response and toxicity studies thatare well known in the art.

The amount of a therapeutic agent, such as the phthalocyaninedye-targeting molecule conjugate (e.g., IR700-antibody conjugate) thatis administered to a human or veterinary subject will vary dependingupon a number of factors associated with that subject, for example theoverall health of the subject. In some embodiments, an effective amountof the agent can be determined by varying the dosage of the product andmeasuring the resulting therapeutic response, such as the regression ofa tumor. In some embodiments, effective amounts can be determinedthrough various in vitro, in vivo or in situ immunoassays. In someembodiments, the disclosed agents can be administered in a single dose,or in several doses, as needed to obtain the desired response. In someembodiments, the effective amount is dependent on the source applied,the subject being treated, the severity and type of the condition beingtreated, and the manner of administration.

In some embodiments, a therapeutically effective amount is an amount ofa composition that alone, or together with an additional therapeuticagent, such as a chemotherapeutic agent, is sufficient to achieve adesired effect in a subject, or in a cell, being treated with thecomposition. The effective amount of the therapeutic agent, such as thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) can be dependent on several factors, including, but notlimited to the subject or cells being treated, the particulartherapeutic agent, and the manner of administration of the therapeuticcomposition. In some embodiments, a therapeutically effective amount orconcentration is one that is sufficient to prevent advancement, such asmetastasis, delay progression, or to cause regression of a disease, orwhich is capable of reducing symptoms caused by the disease, such ascancer. In some embodiments, a therapeutically effective amount orconcentration is one that is sufficient to increase the survival time ofa patient with a tumor.

In some embodiments, a therapeutically effective dose of the conjugateis between or between about 10 mg/m² and 5000 mg/m², such as between orbetween about 10 mg/m² and 3000 mg/m², 10 mg/m² and 1500 mg/m², 10 mg/m²and 750 mg/m², 10 mg/m² and 500 mg/m², 10 mg/m² and 250 mg/m², 10 mg/m²and 200 mg/m², 10 mg/m² and 100 mg/m², 10 mg/m² and 75 mg/m², 10 mg/m²and 50 mg/m², 10 mg/m² and 25 mg/m², 25 mg/m² and 5000 mg/m², 25 mg/m²and 3000 mg/m², 25 mg/m² and 1500 mg/m², 25 mg/m² and 750 mg/m², 25mg/m² and 500 mg/m², 25 mg/m² and 250 mg/m², 25 mg/m² and 200 mg/m², 25mg/m² and 100 mg/m², 25 mg/m² and 75 mg/m², 25 mg/m² and 50 mg/m², 50mg/m² and 5000 mg/m², 50 mg/m² and 3000 mg/m², 50 mg/m² and 1500 mg/m²,50 mg/m² and 750 mg/m², 50 mg/m² and 500 mg/m², 50 mg/m² and 250 mg/m²,50 mg/m² and 200 mg/m², 50 mg/m² and 100 mg/m², 50 mg/m² and 75 mg/m²,75 mg/m² and 5000 mg/m², 75 mg/m² and 3000 mg/m², 75 mg/m² and 1500mg/m², 75 mg/m² and 1000 mg/m², 75 mg/m² and 750 mg/m², 75 mg/m² and 500mg/m², 75 mg/m² and 250 mg/m², 75 mg/m² and 225 mg/m², 75 mg/m² and 200mg/m², 75 mg/m² and 100 mg/m², 100 mg/m² and 5000 mg/m², 100 mg/m² and3000 mg/m², 100 mg/m² and 1500 mg/m², 100 mg/m² and 750 mg/m², 100 mg/m²and 500 mg/m², 100 mg/m² and 250 mg/m², 100 mg/m² and 200 mg/m², 100mg/m² and 150 mg/m², 150 mg/m² and 5000 mg/m², 150 mg/m² and 3000 mg/m²,150 mg/m² and 1500 mg/m², 150 mg/m² and 750 mg/m², 150 mg/m² and 500mg/m², 150 mg/m² and 250 mg/m², 150 mg/m² and 200 mg/m², 200 mg/m² and5000 mg/m², 200 mg/m² and 3000 mg/m², 200 mg/m² and 1500 mg/m², 200mg/m² and 750 mg/m², 200 mg/m² and 500 mg/m², 200 mg/m² and 250 mg/m²,250 mg/m² and 5000 mg/m², 250 mg/m² and 3000 mg/m², 250 mg/m² and 1500mg/m², 250 mg/m² and 750 mg/m², 250 mg/m² and 500 mg/m², 500 mg/m² and5000 mg/m², 500 mg/m² and 3000 mg/m², 500 mg/m² and 1500 mg/m², 500mg/m² and 750 mg/m², 750 mg/m² and 5000 mg/m², 750 mg/m² and 3000 mg/m²,750 mg/m² and 1500 mg/m², 1500 mg/m² and 5000 mg/m², 1500 mg/m² and 3000mg/m², and 3000 mg/m² and 5000 mg/m². In some embodiments, thetherapeutically effective dose of the conjugate is no more than 10mg/m², 50 mg/m², 75 mg/m², 100 mg/m², 150 mg/m², 200 mg/m², 225 mg/m²,250 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m², 800mg/m², 900 mg/m², 1000 mg/m², 1250 mg/m², 1500 mg/m², 2000 mg/m², 2500mg/m², 3000 mg/m², 3500 mg/m², 4000 mg/m², 4500 mg/m², or 5000 mg/m². Insome embodiments, the dose is from or from about 50 mg/m² to about 5000mg/m², from about 250 mg/m² to about 2500 mg/m², from about 750 mg/m² toabout 1250 mg/m² or from about 100 mg/m² to about 1000 mg/m². In someembodiments, the dose is or is about 160 mg/m², 320 mg/m², 640 mg/m² or1280 mg/m².

In some embodiments, a therapeutically effective dose of the conjugateis between or between about 0.25 mg/kg and 150 mg/kg, 0.25 mg/kg and 100mg/kg, 0.25 mg/kg and 75 mg/kg, 0.25 mg/kg and 60 mg/kg, 0.25 mg/kg and50 mg/kg, 0.25 mg/kg and 25 mg/kg, 0.25 mg/kg and 10 mg/kg, 0.25 mg/kgand 7.5 mg/kg, 0.25 mg/kg and 5.0 mg/kg, 0.25 mg/kg and 2.5 mg/kg, 0.25mg/kg and 1.0 mg/kg, 0.25 mg/kg and 0.5 mg/kg, 0.50 mg/kg and 150 mg/kg,0.50 mg/kg and 100 mg/kg, 0.50 mg/kg and 75 mg/kg, 0.50 mg/kg and 60mg/kg, 0.50 mg/kg and 50 mg/kg, 0.50 mg/kg and 25 mg/kg, 0.50 mg/kg and10 mg/kg, 0.50 mg/kg and 7.5 mg/kg, 0.50 mg/kg and 5.0 mg/kg, 0.50 mg/kgand 2.5 mg/kg, 0.50 mg/kg and 1.0 mg/kg, 1.0 mg/kg and 150 mg/kg, 1.0mg/kg and 100 mg/kg, 1.0 mg/kg and 75 mg/kg, 1.0 mg/kg and 60 mg/kg, 1.0mg/kg and 50 mg/kg, 1.0 mg/kg and 25 mg/kg, 1.0 mg/kg and 10 mg/kg, 1.0mg/kg and 7.5 mg/kg, 1.0 mg/kg and 5.0 mg/kg, 1.0 mg/kg and 2.5 mg/kg,2.5 mg/kg and 150 mg/kg, 2.5 mg/kg and 100 mg/kg, 2.5 mg/kg and 75mg/kg, 2.5 mg/kg and 60 mg/kg, 2.5 mg/kg and 50 mg/kg, 2.5 mg/kg and 25mg/kg, 2.5 mg/kg and 10 mg/kg, 2.5 mg/kg and 7.5 mg/kg, 2.5 mg/kg and5.0 mg/kg, 5.0 mg/kg and 150 mg/kg, 5.0 mg/kg and 100 mg/kg, 5.0 mg/kgand 75 mg/kg, 5.0 mg/kg and 60 mg/kg, 5.0 mg/kg and 50 mg/kg, 5.0 mg/kgand 25 mg/kg, 5.0 mg/kg and 10 mg/kg, 5.0 mg/kg and 7.5 mg/kg, 7.5 mg/kgand 150 mg/kg, 7.5 mg/kg and 100 mg/kg, 7.5 mg/kg and 75 mg/kg, 7.5mg/kg and 60 mg/kg, 7.5 mg/kg and 50 mg/kg, 7.5 mg/kg and 25 mg/kg, 7.5mg/kg and 10 mg/kg, 10 mg/kg and 150 mg/kg, 10 mg/kg and 100 mg/kg, 10mg/kg and 75 mg/kg, 10 mg/kg and 60 mg/kg, 10 mg/kg and 50 mg/kg, 10mg/kg and 25 mg/kg, 25 mg/kg and 150 mg/kg, 25 mg/kg and 100 mg/kg, 25mg/kg and 75 mg/kg, 25 mg/kg and 60 mg/kg, 25 mg/kg and 50 mg/kg, 50mg/kg and 150 mg/kg, 50 mg/kg and 100 mg/kg, 50 mg/kg and 75 mg/kg, 50mg/kg and 60 mg/kg, 60 mg/kg and 150 mg/kg, 60 mg/kg and 100 mg/kg, 60mg/kg and 75 mg/kg, 75 mg/kg and 150 mg/kg, 75 mg/kg and 100 mg/kg, and100 mg/kg and 150 mg/kg. n some embodiments, the therapeuticallyeffective dose of the conjugate is no more than 0.25 mg/kg, 0.5 mg/kg,1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90mg/kg, 100 mg/kg, 125 mg/kg or 150 mg/kg.

In some embodiments, the therapeutically effective amount is at least orat least about 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100mg, 200 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 2000 mg,3000 mg or more.

In some embodiments, the methods include administering to a subjecthaving a disease or condition a therapeutically effective amount of aphthalocyanine dye-targeting molecule conjugate, e.g., IR700-antibodyconjugate. In some embodiments, the phthalocyanine dye-targetingmolecule conjugate is targeted to a cell present in the microenvironmentof a tumor, lesion or hyperplasia. In some embodiments, atherapeutically effective dose of the conjugate is administeredintravenously. In some embodiments, a therapeutically effective dose ofthe conjugate is administered intratumorally.

In some embodiments, the dose of the conjugate is at least 10 μg/kg,such as at least 100 μg/kg, at least 500 μg/kg, or at least 500 μg/kg,for example 10 μg/kg to 1000 μg/kg, such as a dose of about 100 μg/kg,about 250 μg/kg, about 500 μg/kg, about 750 μg/kg, or about 1000 μg/kg,for example when administered intratumorally or intraperitoneally (IP).In some embodiments, the dose is at least 1 μg/ml, such as at least 500μg/ml, such as between 20 μg/ml to 100 μg/ml, such as about 10 μg/ml,about 20 μg/ml, about 30 μg/ml, about 40 μg/ml, about 50 μg/ml, about 60μg/ml, about 70 μg/ml, about 80 μg/ml, about 90 μg/ml or about 100μg/ml, for example administered in topical solution.

In some embodiments, the therapeutically effective dose is a doseadministered to a human. In some embodiments, the weight of an averagehuman is 60 to 85 kg, such as about or approximately 75 kg.

In some embodiments, a therapeutically effective dose is one in which anadministered conjugate containing a phthalocyanine dye conjugated to atargeting molecule (e.g., antibody or antigen-binding antibody fragment)achieves a systemic exposure that is no more than the therapeuticallyeffective systemic exposure of the targeting molecule (e.g., antibody orantigen-binding antibody fragment) that is not so conjugated, such asoccurs upon administration of a clinically acceptable dose of the drugtargeting molecule drug alone.

The term “systemic exposure” refers to the actual body exposure of adrug targeting molecule in the plasma (blood or serum) afteradministration of the drug targeting molecule, and can be set forth asthe area under the plasma drug concentration-time curve (AUC) asdetermined by pharmacokinetic analysis after administration of a dose ofthe drug targeting molecule. In some cases, the AUC is expressed inmg*h/L or in corresponding units thereof (e.g., μg*h/L). In someembodiments, the AUC is measured as an average AUC in a patientpopulation, such as a sample patient population, e.g., the average AUCfrom one or more patient(s). In some embodiments, systemic exposurerefers to the area under the curve (AUC) from 0 to infinity (inf or ∞)(AUC_(0-∞) or AUC[0-inf]), including all measured data and dataextrapolated from measured pharmacokinetic (PK) parameters, such as anaverage AUC from a patient population, such as a sample patientpopulation. In some embodiments, AUC_(0-∞) is predicted based on PKinformation for one month. In some embodiments, systemic exposure refersto the AUC from 0 to the last time-point that is experimentally measured(AUC_(0-last)). In some embodiments, the systemic exposure is theexposure (AUC) that occurs at the time of light irradiation orillumination, since the PIT depends on the dose or amount of theconjugate in the tumor at the time when the illumination or irradiationis carried out. In some embodiments, light irradiation or illuminationis carried out within or about within or about 24 hours±3 hours, such as24 hours±2 hours after administration of the conjugate. Thus, in someembodiments, systemic exposure refers to the area under the curve (AUC)from 0 to 24 hours (AUC₀₋₂₄ or AUC[0-24]). In some embodiments, systemicexposure refers to the average area under the curve (AUC) from 0 to 24hours (AUC₀₋₂₄ or AUC[0-24]) from a patient population, such as a samplepatient population.

In some embodiments, the therapeutically effective dose is one in whichan administered conjugate containing a phthalocyanine dye conjugated toa targeting molecule (e.g., antibody or antigen-binding antibodyfragment) achieves a systemic exposure as measured based on AUC₀₋₂₄ thatis substantially lower than the AUC_(0-∞) of a clinically acceptabledose of the targeting molecule that is not so conjugated. In some cases,this is because a therapeutically effective systemic exposure of theconjugate achieved at the time of PIT (at the time of light irradiationor illumination) is the relevant period for the PIT activity asdescribed above. In contrast, in some cases, for a therapeuticallyeffective systemic exposure, such as to achieve pharmacologicalactivity, for a targeting molecule that is not so conjugated (e.g.unconjugated cetuximab), the targeting molecule (e.g. antibody) must bepresent at a much higher exposure. For example, under the FDA guidelinesfor Erbitux (cetuximab), the antibody is administered at 400 mg/m² withweekly doses of 250 mg/m². In some cases, patients may require thecontinuous treatment of an unconjugated targeting molecule (e.g.antibody) for more than one month.

The term “therapeutically effective systemic exposure” refers to thesystemic exposure achieved by a dose of a drug targeting molecule (e.g.,antibody) for pharmacological activity that is deemed to be clinicallyacceptable and/or that achieves a therapeutic effect while having anacceptable safety profile. It is within the level of a skilled artisanto determine or identify a dose of a drug targeting molecule (e.g.,antibody) that is clinically acceptable and/or that achieves atherapeutic effect having an acceptable safety profile. In someembodiments, a clinically acceptable dose of a drug targeting moleculeis determined as the result of clinical trials in animals, andparticularly humans, such as performed by the Food and DrugAdministration (FDA) or other regulatory agencies (e.g. EMA, PDMA). Insome embodiments, a therapeutically effective systemic exposure includesthe systemic exposure resulting either from single dosage administrationof a drug targeting molecule (e.g., antibody) or by repeatedadministration of a drug targeting molecule (e.g., agent) in a cycle ofadministration, such as daily, weekly, biweekly or monthly dosing.

Exemplary FDA approved clinically acceptable dosing schedules forexemplary antibody drugs are set forth in Table 1. In some embodiments,a therapeutically effective systemic exposure of a drug targetingmolecule, including a drug targeting molecule that is not conjugated toa phthalocyanine dye, can be determined or is known from pharmacokineticstudies of a population of subjects at the clinically acceptable orapproved dose administered as either a single administration of theinitial dose or administered by repeated administrations in a dosagecycle (see e.g., Fracasso et al. (2007) Clin. Cancer. Res., 13:986 forobserved systemic exposures (AUC) following single dosage administrationof doses of cetuximab within the dosing range approved by the FDA).

TABLE 1 FDA Approved Doses of Exemplary Drug Antibody Targetingmolecules Exemplary Antibody Therapeutic Dose Indication Cetuximab 400mg/m² (~10 mg/kg) Head and Neck Cancer (Erbitux ®) followed by weeklydose of Colorectal Cancer 250 mg/m² (~6.75 mg/kg) In combination withradiation therapy or platinum-based therapy with 5-FU Bevacizumab 5mg/kg (~185 mg/m²) IV Metastatic colorectal (Avastin ®) every 2 weekswith bolus-IFL cancer 10 mg/kg (~370 mg/m²) IV every 2 weeks withFOLFOX4 5 mg/kg (~185 mg/m²) IV every 2 weeks or 7.5 mg/kg IV every 3weeks with fluoropyrimidine- irinotecan or fluoropyrimidine-oxaliplatinbased chemotherapy 15 mg/kg (~185 mg/m²) Non-squamous IV every 3 weeksnon-small with carboplatin/paclitaxel cell lung cancer 10 mg/kg (~370mg/m²) IV Glioblastoma every 2 weeks 10 mg/kg (~370 mg/m²) IV Metastaticrenal cell every 2 weeks carcinoma with interferon alfa Panitumumab 6mg/kg (~220 mg/m²) metastatic colorectal (Vectibix ®) every 14 dayscancer Retuximab 375 mg/m2 (~10 mg/kg) Non-Hodgkin's (Rituxan ®)Lymphoma (NHL) 375 mg/m2 (~10 mg/kg) in Chronic Lymphocytic the firstcycle and Leukemia (CLL) 500 mg/m2 ((~13 mg/kg) In cycles 2-6, incombination with FC, administered every 28 days Alemtuzumab 30 mg/daythree times B-cell chronic (Campath ®) per week for 12 weeks lymphocyticleukemia (B-CLL) Novolumab 3 mg/kg (~111 mg/m²) metastatic non- (Opdivointravenously small cell lung Injection) every two weeks cancer (NSCLC)pembrolizumab 2 mg/kg (~74 mg/m²) metastatic non-small (KEYTRUDA)administered as an cell lung cancer intravenous infusion over (NSCLC) 30minutes every 3 weeks obtained fromwww.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm

In some embodiments, the therapeutically effective dose of the conjugateis one in which the administered conjugate containing a phthalocyaninedye (e.g., IR700) conjugated to a targeting molecule (e.g., antibody orantigen-binding antibody fragment) achieves an average systemic exposure(e.g., AUC) for a patient population, such as a sample patientpopulation, that no more than 75%, no more than 70%, no more than 60%,no more than 50%, no more than 45%, no more than 40%, no more than 35%,no more than 25%, no more than 20% of the therapeutically averageeffective systemic exposure for a patient population, such as a samplepatient population of the corresponding targeting molecule (e.g.,antibody or antigen-binding antibody fragment) that is not soconjugated. Typically, the systemic exposure of the conjugate issufficiently high to be capable of exhibiting phototoxicity by PIT.

In some embodiments, the conjugate is administered to achieve an averagesystemic exposure as measured by the area under the plasma conjugateconcentration-time curve from time 0 to infinity (AUC[0-inf] orAUC_(0-∞)) for patient population, such as a sample patient population,after administration of the conjugate is between or between about 250μg/mL*h (used interchangeably with μg*h/mL) and 100,000 μg/mL*h, betweenor between about 500 μg/mL*h and 50,000 μg/mL*h, between or aboutbetween 500 μg/mL*h and 25,000 μg/mL*h, between or between about 500μg/mL*h and 18,000 μg/mL*h, between or between about 500 μg/mL*h and10,000 μg/mL*h, between or between about 500 μg/mL*h and 5,000 μg/mL*hor between or between about 500 μg/mL*h and 2,500 μg/mL*h. In someembodiments, the conjugate is administered to achieve a systemicexposure as measured by the average area under the plasma conjugateconcentration-time curve from time 0 to infinity (AUC[0-inf] orAUC_(0-∞)) for patient population, such as a sample patient population,after administration of the conjugate that is no more than 100,000μg/mL*h, no more than 75,000 μg/mL*h, no more than 50,000 μg/mL*h, nomore than 40,000 μg/mL*h, no more than 30,000 μg/mL*h, no more than20,000 μg/mL*h, no more than 10,000 μg/mL*h, no more than 5,000 μg/mL*h,no more than 2,500 μg/mL*h.

In some embodiments, the conjugate is administered to achieve an averagesystemic exposure as measured by the area under the plasma conjugateconcentration-time curve from time 0 to 24 hours (AUC[0-24] or AUC₀₋₂₄)for patient population, such as a sample patient population, afteradministration of the conjugate that is between or between about 100μg/mL*h and 25,000 μg/mL*h, between or between about 200 μg/mL*h and10,000 μg/mL*h, between or between about 500 μg/mL*h and 5,000 μg/mL*h;or the average systemic exposure as measured by AUC₀₋₂₄ for patientpopulation, such as a sample patient population, after administration ofthe conjugate is no more than 25,000 μg/mL*h, no more than 15,000μg/mL*h, no more than 10,000 μg/mL*h, no more than 5,000 μg/mL*h, nomore than 2,500 μg/mL*h, no more than 1,000 μg/mL*h, or no more than 500μg/mL*h. In some embodiments, the plasma conjugate AUC₀₋₂₄ afteradministration of the conjugate is between or between about 500 μg/mL*hand 8,000 μg/mL*h, between or between about 500 μg/mL*h and 5,000μg/mL*h, between or between about 500 μg/mL*h and 2,000 μg/mL*h orbetween or between about 1000 μg/mL*h and 4,000 μg/mL*h.

In some embodiments, the therapeutically effective dose of thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) is less than the single administration therapeuticallyeffective dose of the corresponding targeting molecule (e.g., antibodyor antigen-binding antibody fragment) that is not so conjugated, such asis no more than 75%, no more than 70%, no more than 60%, no more than50%, no more than 45%, no more than 40%, no more than 35%, no more than25%, no more than 20% of the single administration therapeuticallyeffective dose of the corresponding targeting molecule that is not soconjugated.

In some embodiments, the therapeutically effective dose of thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) is less than the initial dose in a repeated dosage scheduleof the therapeutically effective dose of the corresponding targetingmolecule (e.g., antibody or antigen-binding antibody fragment) that isnot so conjugated, such as is no more than 75%, no more than 70%, nomore than 60%, no more than 50%, no more than 45%, no more than 40%, nomore than 35%, no more than 25%, no more than 20% of the initial dose ina repeated dosage administration of the therapeutically effective doseof the corresponding targeting molecule that is not so conjugated.

In some embodiments, the therapeutically effective dose of thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) is less than the average dose of a repeated dosage scheduleof the therapeutically effective dose of the corresponding targetingmolecule (e.g., antibody or antigen-binding antibody fragment) that isnot so conjugated, such as is no more than 75%, no more than 70%, nomore than 60%, no more than 50%, no more than 45%, no more than 40%, nomore than 35%, no more than 25%, no more than 20% of the average dose ina repeated dosage schedule of the therapeutically effective dose of thecorresponding targeting molecule that is not so conjugated.

In some embodiments, a therapeutically effective dose of the conjugateis less than 400 mg/m², less than 300 mg/m², less than 250 mg/m², lessthan 225 mg/m², less than 200 mg/m², less than 180 mg/m², less than 100mg/m² or less than 50 mg/m². In some embodiments, a therapeuticallyeffective dose of the conjugate is between or about between 50 mg/m² and400 mg/m², 100 mg/m² and 300 mg/m², 100 mg/m² and 250 mg/m² or 100 mg/m²and 160 mg/m². In some embodiments, a therapeutically effective dose ofthe conjugate is between or between about 80 mg/m² and 240 mg/m², 80mg/m² and 220 mg/m², 80 mg/m² and 200 mg/m², 80 mg/m² and 180 mg/m², 80mg/m² and 160 mg/m², 80 mg/m² and 140 mg/m², 80 mg/m² and 120 mg/m², 80mg/m² and 100 mg/m², 100 mg/m² and 240 mg/m², 100 mg/m² and 220 mg/m²,100 mg/m² and 200 mg/m², 100 mg/m² and 180 mg/m², 100 mg/m² and 160mg/m², 100 mg/m² and 140 mg/m², 100 mg/m² and 120 mg/m², 120 mg/m² and240 mg/m², 120 mg/m² and 220 mg/m², 120 mg/m² and 200 mg/m², 120 mg/m²and 180 mg/m², 120 mg/m² and 160 mg/m², 120 mg/m² and 140 mg/m², 140mg/m² and 240 mg/m², 140 mg/m² and 220 mg/m², 140 mg/m² and 200 mg/m²,140 mg/m² and 180 mg/m², 140 mg/m² and 160 mg/m², 160 mg/m² and 240mg/m², 160 mg/m² and 220 mg/m², 160 mg/m² and 200 mg/m², 160 mg/m² and180 mg/m², 180 mg/m² and 240 mg/m², 180 mg/m² and 220 mg/m², 180 mg/m²and 200 mg/m², 200 mg/m² and 220 mg/m² or 200 mg/m² and 240 mg/m².

In some embodiments, a therapeutically effective dose of the conjugateis less than 12 mg/kg, less than 10 mg/kg, less than 8 mg/kg, less than6 mg/kg, less than 4 mg/kg, less than 2 mg/kg or less than 1 mg/kg. Insome embodiments, a therapeutically effective dose of the conjugate isbetween or between about 1 mg/kg and 12 mg/kg, 2 mg/kg and 10 mg/kg, 2mg/kg and 6 mg/kg or 2 mg/kg and 4 mg/kg. In some embodiments, atherapeutically effective dose of the conjugate is between or betweenabout 2.0 mg/kg and 6.5 mg/kg, 2.0 mg/kg and 6.0 mg/kg, 2.0 mg/kg and5.0 mg/kg, 2.0 mg/kg and 4.0 mg/kg, 2.0 mg/kg and 3.0 mg/kg, 3.0 mg/kgand 6.5 mg/kg, 3.0 mg/kg and 6.0 mg/kg, 3.0 mg/kg and 5.0 mg/kg, 3.0mg/kg and 4.0 mg/kg, 4.0 mg/kg and 6.5 mg/kg, 4.0 mg/kg and 6.0 mg/kg,4.0 mg/kg and 5.0 mg/kg, 5.0 mg/kg and 6.5 mg/kg, 5.0 mg/kg and 6.0mg/kg and 6.0 mg/kg and 6.5 mg/kg.

In some embodiments, the therapeutically effective amount is betweenabout 75 mg and 500 mg, 75 mg and 400 mg, 75 mg and 400 mg, 75 mg and300 mg, 75 mg and 200 mg, 75 mg and 150 mg, 150 mg and 500 mg, 150 mgand 400 mg, 150 mg and 300 mg, 150 mg and 200 mg, 200 mg and 500 mg, 200mg and 400 mg, 200 mg and 300 mg, 300 mg and 500 mg, 300 mg and 400 mgor 400 mg and 500 mg.

In some embodiments, the conjugate is IR700-cetuximab. In someembodiments, the therapeutically effective amount of IR700-cetuximabconjugate is at least or about at least or is or is about 160 mg/m², 320mg/m² or 640 mg/m². In some embodiments, the therapeutically effectiveamount of IR700-cetuximab conjugate is at least or about at least or isor is about 4.3 mg/kg, 8.6 mg/kg or 17 mg/kg.

In some embodiments, the therapeutically effective dose of the conjugateis for single dosage administration. In some embodiments, thetherapeutically effective dose is administered as only a singleinjection or a single infusion in a dosage schedule or cycle, forexample, is administered only one time in a dosage schedule or cycle.For example, in a dosing schedule or cycle, a subsequent dose of theconjugate is not administered. In some embodiments, the dosing schedulecan be repeated. In some embodiments, the repeated dose, such asrepeated single dose, is administered at a time in which the first dosehas been cleared from the subject, which, in some cases, is a time atwhich there is no detectable systemic exposure of the conjugate. Thus,in some embodiments, the dosing of the conjugate is not administered toachieve a continuous systemic exposure of the conjugate, which isdifferent than many existing therapies, including antibody therapies, inwhich repeating dosing in a dosing schedule or cycle is required tomaintain continuous systemic exposure. In some embodiments, the dosingschedule or cycle is repeated once a week, every two weeks, once amonth, twice a year, once a year or at a lesser frequency as needed.

In some embodiments, in any of the methods for treating provided herein,the dosing schedule is repeated, if residual lesion remains after aprior treatment with the conjugate. In some embodiments, the methodadditionally includes assessing the subject for the presence of aresidual lesion and if residual lesion remains repeating the dosingschedule. In some embodiments, the dosing schedule is repeated if aresidual lesion remains at a time that is more than or about or 1 week,2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after initiationof the prior administration of the conjugate. In some embodiments, thedosing schedule is repeated if a residual lesion remains at or about 4weeks after initiation of the prior administration of the conjugate.

In some embodiments, in a dosing schedule or cycle, a subsequent dose ofthe targeting molecule, e.g., therapeutic targeting molecules (e.g.,therapeutic antibodies) that are not so conjugated to a photosensitizer(e.g., IR700), is not administered. For example, in some embodiments, adose of the phthalocyanine dye-targeting molecule conjugate is notfollowed by a dose of the targeting molecule alone.

One skilled in the art will recognize that higher or lower dosages ofthe phthalocyanine dye-targeting molecule conjugate can also be used,for example depending on the particular agent. In some embodiments,dosages, such as daily dosages, are administered in one or more divideddoses, such as 2, 3, or 4 doses, or in a single formulation. Thephthalocyanine dye-targeting molecule conjugate can be administeredalone, in the presence of a pharmaceutically acceptable carrier, or inthe presence of other therapeutic agents, such as an immune-modulatingagent, anti-cancer agent or other anti-neoplastic agents.

In some embodiments, the phthalocyanine dye-targeting molecule conjugatemay be administered either systemically or locally to the organ ortissue to be treated. Exemplary routes of administration include, butare not limited to, topical, injection (such as subcutaneous,intramuscular, intradermal, intraperitoneal, intratumoral, andintravenous), oral, sublingual, rectal, transdermal, intranasal, vaginaland inhalation routes. In some embodiments, the phthalocyaninedye-targeting molecule conjugate is administered intravenously. In someembodiments, the phthalocyanine dye-targeting molecule conjugate isadministered parenterally. In some embodiments, the phthalocyaninedye-targeting molecule conjugate is administered enterally. In someembodiments, the conjugate is administered by local injection. In someembodiments, the conjugate is administered as a topical application.

The compositions comprising the phthalocyanine dye-targeting moleculeconjugate can be administered locally or systemically using any methodknown in the art, for example to subjects having a tumor, such as acancer, or who has had a tumor previously removed, for example viasurgery. Although specific examples are provided, one skilled in the artwill appreciate that alternative methods of administration of thedisclosed agents can be used. Such methods may include for example, theuse of catheters or implantable pumps to provide continuous infusionover a period of several hours to several days into the subject in needof treatment.

In some embodiments, the phthalocyanine dye-targeting molecule conjugateis administered by parenteral means, including direct injection orinfusion into a tumor, such as intratumorally. In some embodiments, thephthalocyanine dye-targeting molecule conjugate is administered to thetumor by applying the agent to the tumor, for example by bathing thetumor in a solution containing the agent, such as the phthalocyaninedye-targeting molecule conjugate, or by pouring the agent onto thetumor.

In addition, or alternatively, the disclosed compositions can beadministered systemically, for example intravenously, intramuscularly,subcutaneously, intradermally, intraperitoneally, subcutaneously, ororally, to a subject having a tumor, such as cancer.

The dosages of the phthalocyanine dye-targeting molecule conjugate to beadministered to a subject are not subject to absolute limits, but willdepend on the nature of the composition and its active ingredients andits unwanted side effects, such as immune response against the agent,the subject being treated, and the type of condition being treated andthe manner of administration. Generally, the dose will be atherapeutically effective amount, such as an amount sufficient toachieve a desired biological effect, for example an amount that iseffective to decrease the size, such as volume and/or weight, of thetumor, or attenuate further growth of the tumor, or decrease undesiredsymptoms of the tumor.

In some embodiments, the compositions used for administration of theagent, such as the phthalocyanine dye-targeting molecule conjugatecontain an effective amount of the agent along with conventionalpharmaceutical carriers and excipients appropriate for the type ofadministration contemplated. For example, in some embodiments,parenteral formulations may contain a sterile aqueous solution orsuspension of the conjugate. In some embodiments, compositions forenteral administration may contain an effective amount of thephthalocyanine dye-targeting molecule conjugate in aqueous solution orsuspension that may optionally include buffers, surfactants, thixotropicagents, and flavoring agents.

C. Dosage Regime and Photoimmunotherapy

The PIT includes administration of a composition containing thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate) followed by irradiation. In some embodiments, the methodincludes irradiating the tumor.

In some embodiments, after the cells are contacted with thephthalocyanine dye-targeting molecule conjugate, the cells areirradiated. Methods of irradiation are known in the art. As only cellsexpressing the cell surface protein will typically be recognized by thetargeting molecule, generally only those cells will have sufficientamounts of the conjugate bound to it. This may decrease the likelihoodof undesired side effects, such as killing of normal cells, as theirradiation may only kill the cells to which the conjugate is bound, andgenerally not other cells.

In some embodiments, a cell is irradiated in vivo, for exampleirradiating a subject who has previously been administered thephthalocyanine dye-targeting molecule conjugate. In some embodiments,the subject is irradiated, for example a tumor in the subject can beirradiated.

In some embodiments, the irradiation is effected after administration ofthe phthalocyanine dye-targeting molecule conjugate. In someembodiments, the irradiation or illumination is carried out or effectedbetween or between about 30 minutes and 96 hours after administering thephthalocyanine dye-targeting molecule conjugate (e.g., IR700-antibodyconjugate), such as between 30 minutes and 48 hours, 30 minutes and 24hours or 12 hours and 48 hours, such as generally at least 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours or more after administering the conjugate. For example,the irradiation can be performed within about 24 hours afteradministering the conjugate. In some embodiments, greater than 6 hoursprior to irradiating or illuminating the tumor, the subject has beenadministered the conjugate comprising the targeting molecule, whereinthe conjugate associates with the tumor. In some embodiments, theconjugate has been previously administered to the subject greater thanor greater than about 12 hours, 24 hours, 26 hours, 48 hours, 72 hoursor 96 hours prior to irradiating or illuminating the tumor.

In some embodiments, at the time of or after the irradiation, thesubject can receive one or more other therapies (e.g., immune-modulatingagent or anti-cancer agent) as described herein. In some cases, the oneor more other therapies are thus also administered after administrationof the phthalocyanine dye-targeting molecule conjugate (e.g.,IR700-antibody conjugate). In some embodiments, the additional therapyis administered within or within about 0 to 24 hours of the irradiation,such as within or within about 5 minutes, 10 minutes, 30 minutes, 1hour, 2 hours, 6 hours, 12 hours or 24 hours of the irradiation.

In some embodiments, prior to the irradiation, the subject can receiveone or more other therapies as described herein. In some cases, the oneor more other therapies can be administered prior to, during, orfollowing administration of the phthalocyanine dye-targeting moleculeconjugate (e.g., IR700-antibody conjugate), and generally prior toirradiation of the subject. In some embodiments, the additionaltherapeutic agent (e.g., immune-modulating agent or anti-cancer agent)can be administered during or simultaneously with administration of thephthalocyanine dye-targeting molecule conjugate. In some embodiments,the additional therapeutic agent (e.g., immune-modulating agent oranti-cancer agent) can be administered after or following administrationof the phthalocyanine dye-targeting molecule conjugate. For example, insome embodiments, the conjugate is administered prior to the one or moreother therapies and the conjugate and one or more other therapies areeach administered prior to irradiating the tumor. In some embodiments,the conjugate is administered subsequent to the one or more othertherapies and the conjugate and one or more other therapies are eachadministered prior to irradiating the tumor. In some embodiments, theirradiation is carried out after administration of the additionaltherapeutic (e.g., immune modulating agent or anti-cancer agent) and thephthalocyanine dye-targeting molecule conjugate.

In some embodiments, the additional therapeutic agent is an immunemodulating agent and the immune modulating agent is administered 6 hoursto 4 weeks prior to the irradiation, such as generally greater than orgreater than about 12 hours, 24 hours, 36 hours, 72 hours, 96 hours, oneweek, two weeks, three weeks or four weeks prior to the irradiation. Insome embodiments, the phthalocyanine dye-targeting molecule (e.g. IR700antibody conjugate) is administered 6 hours to 96 hours prior to theirradiation, such as generally within or within about or about 6 hours,12 hours, 24 hours, 36 hours, 72 hours or 96 hours prior to theirradiation.

In some embodiments, prior to the irradiation, the subject can receivean immune modulating agent, such as an immune checkpoint inhibitor. Insome embodiments, the immune modulating agent is generally administeredprior to irradiation of the subject. In some embodiments, the immunemodulating agent is administered between or between about 12 hours and 2months before effecting the irradiation, such as between 12 hours and 1month, 12 hours and 3 weeks, 12 hours and 2 weeks, 12 hours and 1 week,and 1 week and 1 month, such as generally at least 12 hours, 24 hours,48 hours, 96 hours, one week, two weeks, three weeks, or one month priorto irradiating the tumor. In some embodiments, the immune modulatingagent can be administered prior to, subsequent to or simultaneously withthe conjugate, so long as both the immune modulating agent and conjugateare administered prior to the irradiation. In some embodiments, theimmune modulating agent can be administered prior to the administrationof the phthalocyanine dye-targeting molecule conjugate (e.g.,IR700-antibody conjugate). In some embodiments, the immune modulatingagent can be administered during or simultaneously with administrationof the phthalocyanine dye-targeting molecule conjugate. In someembodiments, the immune modulating agent can be administered subsequentto or following administration of the phthalocyanine dye-targetingmolecule conjugate. For example, the immune modulating agent subsequentto the irradiation three times a week, two times a week, once everyweek, once every two weeks, once every three weeks or once a month.

For example, in some cases the conjugate is administered at least 12hours, such as from or from about 12 hours to 48 hours, prior toirradiation and the immune modulating agent is administered at least 12hours, such as from or from about 12 hours to about 1 month, prior toirradiation. In some embodiments, the conjugate is administered no morethan or no more than about 36 hours, 24 hours, 18 hours or 12 hoursprior to irradiation and the immune modulating agent is administeredmore than 12 hours prior to irradiation, such as generally more than 24hours, 48 hours, 96 hours, one week, two weeks, three weeks or one monthprior to irradiation.

In some embodiments, the immune modulating agent is itself a conjugatecontaining a phthalocyanine dye, such as a phthalocyanine dye linked toan antibody or antigen-binding fragment that is an immune modulatingagent. In some embodiments, the immune modulating agent is anIR700-antibody conjugate that includes an immune modulating antibody(e.g. checkpoint inhibitor) that binds to a checkpoint protein on atumor cell (e.g. PD-L1). In some embodiments, the immune modulatingconjugate (e.g., IR700-antibody conjugate that is an immune modulatingagent) is administered prior to administration of the phthalocyaninedye-targeting molecule conjugate, such as between 12 hours and 2 months,such as generally at least 12 hours, at least 24 hours, at least 48hours, at least 96 hours, at least one week, at least two weeks, atleast three weeks or at least one month prior to administration of thephthalocyanine dye-targeting molecule conjugate. In some embodiments,the immune modulating conjugate (e.g., IR700-antibody conjugate that isan immune modulating agent) is administered during or simultaneouslywith administration of the phthalocyanine dye-targeting moleculeconjugate. In some embodiments, the immune modulating conjugate (e.g.,IR700-antibody conjugate that is an immune modulating agent) isadministered after administration of the phthalocyanine dye-targetingmolecule conjugate, such as between 12 hours and 2 months, such asgenerally at least 12 hours, at least 24 hours, at least 48 hours, atleast 96 hours, at least one week, at least two weeks, at least threeweeks or at least one month after administration of the phthalocyaninedye-targeting molecule conjugate.

In some embodiments, the irradiation is carried out or effected afteradministration of the immune modulating conjugate (e.g., IR700-antibodyconjugate that is an immune modulating agent) and the phthalocyaninedye-targeting molecule conjugate. In some embodiments, the irradiationis effected after administration of the phthalocyanine dye-targetingmolecule conjugate.

In some embodiments, the method of combination therapy includes twoirradiations or illuminations. In some embodiments, the method ofcombination therapy involves a first irradiation of the tumor afteradministering the immune modulating conjugate (e.g., IR700-antibodyconjugate that is an immune modulating agent) and a second irradiationof the tumor after administering the phthalocyanine dye-targetingmolecule conjugate. In some embodiments, each irradiation is performedwithin 6 to 48 hours after administering the respective conjugate, suchas generally at least about 6 hours, 12 hours, 24 hours or 36 hoursafter administration of each conjugate.

In some embodiments, the method of combination therapy comprising theimmune modulating conjugate (e.g., IR700-antibody conjugate that is animmune modulating agent) and the phthalocyanine dye-targeting moleculeconjugate only includes a single irradiation. In some embodiments, thephthalocyanine dye-targeting molecule conjugate is administered at least12 hours after administering the immune modulating conjugate (e.g.,IR700-antibody conjugate that is an immune modulating agent) and within6 to 48 hours prior to irradiating the tumor.

In some embodiments, the immune modulating conjugate (e.g.,IR700-antibody conjugate that is an immune modulating agent) and thephthalocyanine dye-targeting molecule conjugate are administered by thesame route of administration. In some embodiments, both the immunemodulating conjugate (e.g., IR700-antibody conjugate that is an immunemodulating agent) and the phthalocyanine dye-targeting moleculeconjugate are administered systemically. In other embodiments, both ofthe conjugates are administered intravenously.

In some embodiments, prior to the irradiation, the subject can receivean anti-cancer agent. In some embodiments, the anti-cancer agent isgenerally administered prior to irradiation of the subject. In someembodiments, the irradiation is carried out or effected between orbetween about 5 minutes and 2 weeks after administering the anti-canceragent, such as between 5 minutes and 1 week, 5 minutes and 3 days, 5minutes and 48 hours, 5 minutes and 24 hours, 5 minutes and 12 hours, 5minutes and 6 hours, and 5 minutes and 1 hour, such as generally atleast about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 12 hours, or 24 hours prior to irradiating the tumor. Insome embodiments, the anti-cancer agent can be administered prior to,subsequent to or simultaneously with the conjugate, so long as both theanti-cancer agent and conjugate are administered prior to theirradiation. In some embodiments, the anti-cancer agent can beadministered prior to the administration of the phthalocyaninedye-targeting molecule conjugate. In some embodiments, the anti-canceragent can be administered during or simultaneously with administrationof the phthalocyanine dye-targeting molecule conjugate. In someembodiments, the anti-cancer agent can be following administration ofthe phthalocyanine dye-targeting molecule conjugate.

For example, in some cases the conjugate is administered at least 12hours, such as from or from about 12 hours to 48 hours, prior toirradiation and the anti-cancer agent is administered at least 5minutes, such as from or from about 5 minutes to 24 hours, prior toirradiation. In some embodiments, the conjugate is administered no morethan or no more than about 36 hours, 24 hours, 18 hours or 12 hoursprior to irradiation and the anti-cancer agent is administered greaterthan 5 minutes prior to irradiation and generally no more than 30minutes, 1 hour, 2 hours, 6 hours, 12 hours or 24 hours prior toirradiation.

In some embodiments, the cells, such as a tumor, are irradiated with atherapeutic dose of radiation at a wavelength within a range from orfrom about 400 nm to about 900 nm, such as from or from about 500 nm toabout 900 nm, such as from or from about 600 nm to about 850 nm, such asfrom or from about 600 nm to about 740 nm, such as from about 660 nm toabout 740 nm, from about 660 nm to about 710 nm, from about 660 nm toabout 700 nm, from about 670 nm to about 690 nm, from about 680 nm toabout 740 nm, or from about 690 nm to about 710 nm. In some embodiments,the cells, such as a tumor, are irradiated with a therapeutic dose ofradiation at a wavelength of 600 nm to 850 nm, such as 660 nm to 740 nm.In some embodiments, the cells, such as a tumor, is irradiated at awavelength of at least or about at least 600 nm, 620 nm, 640 nm, 660 nm,680, nm, 700 nm, 720 nm or 740 nm, such as 690±50 nm, for example about680 nm.

In some embodiments, the cells, such as a tumor, are irradiated at adose of at least 1 J cm⁻², such as at least 10 J cm⁻², at least 30 Jcm⁻², at least 50 J cm⁻², at least 100 J cm⁻², or at least 500 J cm⁻².In some embodiments, the dose of irradiation is from or from about 1 toabout 1000 J cm⁻², from about 1 to about 500 J cm⁻², from about 5 toabout 200 J cm⁻², from about 10 to about 100 J cm⁻², or from about 10 toabout 50 J cm⁻². In some embodiments, the cells, such as a tumor, areirradiated at a dose of at least or at least about 2 J cm⁻², 5 J cm⁻²,10 J cm⁻², 25 J cm⁻², 50 J cm⁻², 75 J cm⁻², 100 J cm⁻², 150 J cm⁻², 200J cm⁻², 300 J cm⁻², 400 J cm⁻², or 500 J cm⁻².

In some embodiments, the cells, such as a tumor, are irradiated orilluminated at a dose of at least 1 J/cm fiber length, such as at least10 J/cm fiber length, at least 50 J/cm fiber length, at least 100 J/cmfiber length, at least 250 J/cm fiber length, or at least 500 J/cm fiberlength. In some embodiments, the dose of irradiation is from or fromabout 1 to about 1000 J/cm fiber length, from about 1 to about 500 J/cmfiber length, from about 2 to about 500 J/cm fiber length, from about 50to about 300 J/cm fiber length, from about 10 to about 100 J/cm fiberlength, or from about 10 to about 50 J/cm fiber length. In someembodiments, the cells, such as a tumor, are irradiated at a dose of atleast or at least about 2 J/cm fiber length, 5 J/cm fiber length, 10J/cm fiber length, 25 J/cm fiber length, 50 J/cm fiber length, 75 J/cmfiber length, 100 J/cm fiber length, 150 J/cm fiber length, 200 J/cmfiber length, 250 J/cm fiber length, 300 J/cm fiber length, 400 J/cmfiber length or 500 J/cm fiber length.

In some embodiments, the dose of irradiation or illumination in a humansubject is from or from about 1 to about 400 J cm⁻², from about 2 toabout 400 J cm⁻², from about 1 to about 300 J cm⁻², from about 10 toabout 100 J cm⁻² or from about 10 to about 50 J cm⁻², from about such asis at least or at least about or is or within or within about or is oris about 10 J cm⁻², at least 30 J cm⁻², at least 50 J cm⁻², at least 100J cm⁻². In some embodiments, the dose of irradiation in a human subjectis from or from about 1 to 300 J/cm fiber length, 10 to 100 J/cm fiberlength or 10 to 50 J/cm fiber length, such as is at least or at leastabout or is or within or within about or is or is about 10 J/cm fiberlength, at least 30 J/cm fiber length, at least 50 J/cm fiber length, atleast 100 J/cm fiber length. In some cases, it is found that a dose ofirradiation in a human subject to achieve PIT can be less than isnecessary for PIT in a mouse. For example, in some cases, 50 J/cm² (50 Jcm⁻²) light dosimetry in an in vivo tumor mouse model is not effectivefor PIT, which is in contrast to what we can be observed in the clinicwith human patients.

In some embodiments, the dose of irradiation following administration ofthe composition comprising the phthalocyanine dye-targeting moleculeconjugate is at least 1 J cm⁻² or 1 J/cm of fiber length at a wavelengthof 660-740 nm, for example, at least 10 J cm⁻² or 10 J/cm of fiberlength at a wavelength of 660-740 nm, at least 50 J cm⁻² or 50 J/cm offiber length at a wavelength of 660-740 nm, or at least 100 J cm⁻² or100 J/cm of fiber length at a wavelength of 660-740 nm, for example 1.0to 500 J cm⁻² or 1.0 to 500 J/cm of fiber length at a wavelength of660-740 nm. In some embodiments, the wavelength is 660-710 nm. In someembodiments, the dose of irradiation following administration of thecomposition comprising the phthalocyanine dye-targeting moleculeconjugate is at least 1.0 J cm⁻² or 1 J/cm of fiber length at awavelength of 680 nm for example, at least 10 J cm⁻² or 10 J/cm of fiberlength at a wavelength of 680 nm, at least 50 J cm⁻² or 50 J/cm of fiberlength at a wavelength of 680 nm, or at least 100 J cm⁻² or 100 J/cm offiber length at a wavelength of 680 nm, for example 1.0 to 500 J cm⁻² or1.0 to 500 J/cm of fiber length at a wavelength of 680 nm. In someembodiments, multiple irradiations are performed, such as at least 2, atleast 3, or at least 4 irradiations, such as 2, 3, 4, 5, 6, 7, 8, 9 or10 separate administrations. Exemplary irradiation after administrationof the conjugates or compositions provided herein include irradiatingthe tumor at a wavelength of 660 nm to 740 nm at a dose of at least 1 Jcm⁻² or 1 J/cm of fiber length.

In some embodiments, a light or laser may be applied to the dyemolecules, such as cells containing the conjugate, for from about 5seconds to about 5 minutes. For example, in some embodiments, the lightor laser is applied for or for about 5, 10, 15, 20, 25, 30, 35, 40, 4550 or 55 seconds, or for within a range between any of two such values,to activate the dye molecules. In some embodiments, the light or laseris applied for or for about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 minutes,or more, or within a range between any two of such values. In someembodiments, the length of time a light or laser is applied can varydepending, for example, on the energy, such as wattage, of the light orlaser. For example, lights or lasers with a lower wattage may be appliedfor a longer period of time in order to activate the dye molecule.

In some embodiments, a light or laser may be applied about 30 minutes toabout 48 hours after administering the conjugate. For example, in someembodiments, the light or laser is applied at or at about 30, 35, 40,45, 50 or 55 minutes after administering the conjugate, or within arange between any two of such values. In some embodiments, the light orlaser is applied at or at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours afteradministering the conjugate, or is administered within a range betweenor between about any two of such values. In some embodiments, the lightor laser is applied for between or between about 1 and 24 hours, such asbetween or between about 1 and 12 hours, 12 and 24 hours, 6 and 12hours, or may be administered more than 24 following administration ofthe conjugate. In some embodiments, the light or laser is applied 36 or48 hours after administering the conjugate.

In some embodiments, cells, or subjects, can be irradiated one or moretimes. Thus, irradiation can be completed in a single day, or may bedone repeatedly on multiple days with the same or a different dosage,such as irradiation at least 2 different times, 3 different times, 4different times 5 different times or 10 different times. In someembodiments, repeated irradiations may be done on the same day, onsuccessive days, or every 1-3 days, every 3-7 days, every 1-2 weeks,every 2-4 weeks, every 1-2 months, or at even longer intervals.

In some embodiments, the dose or method of irradiation differs dependingon the type or morphology of the tumor.

In some embodiments, the lesion is a tumor that is a superficial tumor.In some embodiments, the tumor is less than 10 mm thick. In someembodiments, irradiation is carried out using a microlens-tipped fiberfor surface illumination. In some embodiments, the light irradiationdose is from or from about 5 J/cm⁻² to about 200 J/cm⁻².

In some embodiments, the provided methods include illuminating ansuperficial tumor in a subject with a microlens-tipped fiber for surfaceillumination with a light dose of from or from about 5 J/cm⁻² to about200 J/cm⁻², wherein the tumor is associated with a phototoxic agent thatincludes a targeting molecule bound to a cell surface molecule of thetumor. In some embodiments, the light irradiation dose is or is about 50J/cm⁻².

In some embodiments, the lesion is a tumor that is an interstitialtumor. In some embodiments, the tumor is greater than 10 mm deep or is asubcutaneous tumor. In some embodiments, irradiation is carried outusing cylindrical diffusing fibers that includes a diffuser length of0.5 cm to 10 cm and spaced 1.8±0.2 cm apart. In some embodiments, thelight irradiation dose is from or from about 20 J/cm fiber length toabout 500 J/cm fiber length.

In some embodiments, the provided methods include illuminating aninterstitial tumor in a subject with cylindrical diffusing fibers thatincludes a diffuser length of 0.5 cm to 10 cm and spaced 1.8±0.2 cmapart with a light dose of or about 100 J/cm fiber length or with afluence rate of or about 400 mW/cm, wherein the tumor is associated witha phototoxic agent that includes a targeting molecule bound to a cellsurface molecule of the tumor. In some embodiments, the tumor is greaterthan 10 mm deep or is a subcutaneous tumor. In some embodiments, thecylindrical diffusing fibers are placed in a catheter positioned in thetumor 1.8±0.2 cm apart. In some embodiments, the catheter is opticallytransparent.

In some embodiments, the provided methods include irradiation at one ormore wavelengths. In some examples, after administering the conjugate,the lesion or tumor is irradiated at one or more wavelengths to inducephototoxic activity of the first dye of the conjugate and a fluorescentsignal of the second dye of the conjugate. For example, in methods thatemploy a first and second dye conjugated to a targeting molecule thathave different excitation wavelengths, two different wavelengths can beused for irradiation. In some embodiments, the provided methods includeirradiating the lesion with a single wavelength. In some embodiments,the provided methods include irradiating the lesion at two differentwavelengths, simultaneously or sequentially, wherein one wavelengthinduces the phototoxic activity and the other wavelength induces thefluorescent signal. For example, in some embodiments, the providedmethods include irradiating the lesion at one or more wavelengths thatis from or from about 400 to about 900 nm at a dose of at least 1 J cm⁻²or 1 J/cm of fiber length.

D. Additional Therapeutic Agents and Combination Therapy

In some embodiments, another therapeutic agent, such as an immunemodulating agent or anti-cancer agent is administered in conjunctionwith a photoimmunotherapy agent, such as a phthalocyanine dye conjugate,for example an IR700-antibody conjugate. In some embodiments, thecombination therapy can include administration of a phthalocyanine dyeconjugate, for example an IR700-antibody conjugate, in combination withan anti-cancer agent or immune modulating agent.

In some embodiments, the other or additional agent or agents can beadministered at a sufficient time prior to performing the irradiation sothat a therapeutic effect on treating the tumor is increased. In someembodiments, prior to irradiation in the method of photoimmunotherapy,one or more other therapeutic agents, such as an immune modulating agent(e.g., immune checkpoint inhibitor) or anti-cancer agent (e.g.,antimetabolite), are administered to the subject. In one embodiment, animmune modulating agent can be administered a sufficient time prior tothe irradiation, such as generally at least 12 hours prior to theirradiation, to render the immune system responsive to tumor-associatedagents released upon tumor cell lysis after photoimmunotherapy. Inanother embodiment, an anti-cancer agent can be administered asufficient time prior to the irradiation, such as generally at least 5minutes prior to the irradiation, to achieve systemic availability ofthe anti-cancer agent so that it can be immediately delivered into thetumor upon changes in vascular permeability after photoimmunotherapy.

The one or more other agents, such as an immune modulating agent or ananti-cancer agent, can be administered prior to, simultaneous with,subsequent to or intermittently with the phthalocyanine dye-targetingmolecule conjugate. In some embodiments, the activation of thephthalocyanine dye photosensitizer of the conjugate by irradiation withlight is not effected until a time after the administration of the othertherapeutic agent, such as described herein. In some embodiments, theactivation of the phthalocyanine dye photosensitizer of the conjugate byirradiation with light is carried out before the administration of theother therapeutic agent, such as described herein.

In some embodiments, the combined effect of the photoimmunotherapy incombination with the one or more other agents can be synergisticcompared to treatments involving only photoimmunotherapy with thephthalocyanine dye-targeting molecule conjugate or monotherapy with theother therapeutic agent. In some embodiments, the methods providedherein result in an increase or an improvement in a desired anti-tumortherapeutic effect, such as an increased or an improvement in thereduction or inhibition of one or more symptoms associated with cancer,than photoimmunotherapy or monotherapy alone.

Treatments with a phthalocyanine dye-targeting molecule conjugate, and,optionally, an additional therapeutic immune modulating agent oranti-cancer agent, can each independently be completed in a single day,or may be done repeatedly on multiple days with the same or a differentdosage. Repeated treatments may be done on the same day, on successivedays, or every 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4weeks, every 1-2 months, or at even longer intervals.

In some embodiments, the combination therapy includes administering to asubject a therapeutically effective amount of the immune modulatingagent, such as an immune checkpoint inhibitor. The immune modulatingagent is administered in an amount that is from or from about 0.01 mg to1000 mg, such as at a dose of at least 0.01 mg, 0.1 mg, 1 mg, 10 mg,1000 mg, 2000 mg, 3000 mg or more. In an exemplary embodiment, an immunemodulating agent such as an immune checkpoint inhibitor may beadministered at about 0.3 mg/kg to 10 mg/kg, or the maximum tolerateddose, such as at least 0.5 mg/kg, or at least 1 mg/kg, or at least 2mg/kg, or at least 3 mg/kg, or at least 5 mg/kg, or at least 8 mg/kg. Insome cases, the dose can be administered as a single dose or in aplurality of doses. Alternatively, the immune modulating agent such asan immune checkpoint inhibitor may be administered by an escalatingdosage regimen including administering a first dosage at about 3 mg/kg,a second dosage at about 5 mg/kg, and a third dosage at about 9 mg/kg.Alternatively, the escalating dosage regimen includes administering afirst dosage of immune modulating agent at about 5 mg/kg and a seconddosage at about 9 mg/kg. Another stepwise escalating dosage regimen mayinclude administering a first dosage of immune modulating agent about 3mg/kg, a second dosage of about 3 mg/kg, a third dosage of about 5mg/kg, a fourth dosage of about 5 mg/kg, and a fifth dosage of about 9mg/kg. In another aspect, a stepwise escalating dosage regimen mayinclude administering a first dosage of 5 mg/kg, a second dosage of 5mg/kg, and a third dosage of 9 mg/kg. In some embodiments, particulardosages can be administered twice weekly, once weekly, once every twoweeks, once every three weeks or once a month or more. In some cases,the dosages can be administered over a course of a cycle that can berepeated, such as repeated for one month, two months, three months, sixmonths, 1 year or more.

In some embodiments, the combination therapy includes administering to asubject a therapeutically effective amount of the anti-cancer agent,such as any described herein. In some embodiments, a therapeuticallyeffective dose can be from or from about 0.01 mg to 1000 mg, such as adose of at least 0.01 mg, 0.1 mg, 1 mg, 10 mg, 1000 mg, 2000 mg, 3000 mgor more. In some embodiments, a therapeutically effective dose of theanti-cancer agent is from or from about 0.01 mg/kg to about 50 mg/kg,such as about 0.1 mg/kg to about 20 mg/kg, about 0.1 to about 10 mg/kg,about 0.3 to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg or about0.5 mg/kg to about 1 mg/kg.

In some embodiments, the dose of the immune modulating agent (e.g.immune checkpoint inhibitor) or anti-cancer agent is continued orrepeated in accord with its clinically dosing schedule after PITtreatment. Thus, in some embodiments, in a dose schedule or cycle ofadministration in accord with the provided methods, the phthalocyaninedye conjugate (e.g. IR700 antibody conjugate) can be administered onlyone time, such as in a single dose or infusion, for PIT, whereas theadministration of the immune modulating agent is continued or repeatedmore than one time, such as three times a week, two times a week, once aweek, once every two weeks, once every three weeks or once a monthduring a dosing schedule or cycle of administration. In someembodiments, the dosing schedule or cycle of administration is or isabout 28 days or 4 weeks.

In some embodiments, the other or additional agent or agentsadministered, or the additional agent in a combination therapy, is anunconjugated targeting molecule. In some embodiments, the unconjugatedtargeting molecule is the same or substantially the same targetingmolecule as the targeting molecule of the conjugate. For example, insome embodiments, prior to administration of the conjugate, thetargeting molecule, e.g., an unconjugated antibody that targets aprotein or antigen, is administered to the subject. In some embodiments,the targeting molecule is administered up to 96 hours prior toadministration of the conjugate. In some embodiments, the targetingmolecule is administered at a dose within a range from or from about 10mg/m² to about 500 mg/m². For example, the targeting molecule iscetuximab, and cetuximab is administered to the subject up to 96 hoursprior to administration of the conjugate.

1. Immune Modulating Agents

The present disclosure provides immune modulating agents that can beadministered in combination with PIT methods employing phthalocyaninedye conjugates. Hence, the combination therapy provided herein,including combinations and methods of use thereof, include an immunemodulating agent. In some aspects, an immune modulating agents, orimmunomodulators, are substances that either, directly or indirectly,suppress or activate the body's immune response. For example, immunemodulating agents that stimulate immune response to tumors and/orpathogens may be used in combination with photoimmunotherapy. In someembodiments, the immune modulating agent can include cell-based (e.g.combination treatment with immune cells such as dendritic cells or Tcells) or non-cell based immune modulating agents.

Generally, cancerous cells contain tumor-specific antigens that shouldbe recognized by the immune system. Typically, in an active immunesystem, immune cells, such as cytotoxic T cells, attack and eradicatethese cancerous cells. Under normal physiological conditions, the Tcell-mediated immune response is initiated by antigen recognition by theT cell receptor (TCR) and is regulated by a balance of co-stimulatoryand inhibitory signals (e.g. immune checkpoint proteins). In particular,CD4+ and CD8+ T cells expressing a TCR can become activated uponrecognition of antigenic peptides presented on antigen-presenting cellson major histocompatibility complex (MHC) class I or class II molecules,respectively. In some aspects, activated CD8+ cells, or cytotoxic Tcells, can kill tumor cells expressing the antigen, which can be helpedby the presence of CD4+ T cells. In some embodiments, the immune cell isan antigen presenting cell. In some embodiments, the immune cell is adendritic cell.

In the case of tumors, however, the tumor microenvironment hasmechanisms to suppress the immune system, thereby evading immunerecognition and preventing or reducing killing of tumor cells. Forexample, in some cases, immune checkpoint proteins can be dysregulatedin tumors, thereby resulting in a suppression of the immune response inthe tumor microenvironment as a mechanism of evading the immune system.In some cases, other mechanisms can act to inhibit access of immunecells to tumor antigens, thereby also contributing to the tumors abilityto evade the immune system. The combination therapies provided hereinaddress both of these evasion mechanisms, in order to provide a morerobust immune response against the tumor while also killing tumor cellsby photolytic mechanisms.

In some embodiments of the combination therapy methods provided herein,an immune modulating agent is administered to a subject in order toinhibit immunosuppressive signaling or enhance immunostimulantsignaling. For example, inhibitory checkpoint protein antagonists and/oragonists of co-stimulatory receptors can stimulate a host's endogenousanti-tumor immune response by amplifying antigen-specific T cellresponses. In aspects of the provided methods, photoimmunotherapy alsocan be performed, which can result in the killing of tumor cells,thereby releasing tumor-antigens. By performing photoimmunotherapy incombination with administration of an immune-modulating agent, thesubsequent release of PIT-induced antigens can provide a source ofantigenic stimuli for the T cells whose response has been amplified orstimulated by the immune modulating agent. Thus, in some aspects, theenhanced immune response that is generated upon therapy with an immunemodulating agent is primed and ready to respond to tumor antigens thatare exposed upon lysis of cells after PIT. Thus, in some aspects, thecombination therapies provided herein address the natural evasionmechanisms that can be present in a tumor microenvironment, in order toprovide a more robust immune response against the tumor while alsokilling tumor cells by photolytic mechanisms.

Generally, in the provided methods, PIT-mediated cell killing byirradiation and activation of an administered phthalocyaninedye-conjugate is performed at a time after the immune response has beenstimulated or enhanced following administration of an immune-modulatingagent. In some embodiments, the immune-modulating agent is administereda sufficient time prior to irradiation of an administered phthalocyaninedye-conjugate so that amplification of the T cell response has occurredprior to PIT-induced cell lysis. Hence, generally, the immune-modulatingagent is administered a sufficient time prior to irradiation to betherapeutically effective to amplify a T cell response, such asadministered at least 12 hours, such as generally at least 24 hours, atleast 48 hours, at least 96 hours, at least one week, at least twoweeks, at least three weeks or at least one month prior to performingirradiation to induce PIT-mediated cell killing through activation of anadministered phthalocyanine dye-conjugate (e.g., IR700-targetingmolecule conjugate, such as an IR700-antibody dye conjugate).

In some embodiments, the conjugate is administered prior to,simultaneously or subsequently to administration of theimmune-modulating agent. In some embodiments, the conjugate isadministered after administering the immune modulating agent but priorto irradiating the tumor. In some embodiments, the conjugate isadministered from or from about 12 hours to 48 hours prior toirradiating the tumor and the immune modulating agent is administeredfrom or from about 12 hours to about 1 month prior to irradiating thetumor. In some embodiments, immune modulating agent is administeredgreater than or greater than about 30 minutes, 1 hour, 2 hours, 6 hours,12 hours, 24 hours, 48 hours, 96 hours, one week, two weeks, three weeksor one month prior to irradiating the tumor.

In some embodiments, irradiation the tumor is carried out either i)after administration of the immune modulating agent and afteradministration of the conjugate or ii) only after administration of theconjugate.

Exemplary dosage regimes and schedules for administering an immunemodulating agent, phthalocyanine dye-conjugate (e.g., IR700-targetingmolecule conjugate, such as an IR700-antibody dye conjugate) and forperforming irradiation are described elsewhere herein.

In some embodiments, the combination therapy methods can be performedwith any immune modulating agent that can stimulate, amplify and/orotherwise enhance an anti-tumor immune response, such as by inhibitingimmunosuppressive signaling or enhancing immunostimulant signaling. Insome embodiments, the immune modulating agent is a peptide, protein oris a small molecule. In some embodiments, the protein can be a fusionprotein or a recombinant protein. In some embodiments, the immunemodulating agent binds to an immunologic target, such as a cell surfacereceptor expressed on immune cells, such a T cells, B cells orantigen-presenting cells. For example, in some embodiments, the immunemodulating agent is an antibody or antigen-binding antibody fragment, afusion protein, a small molecule or a polypeptide.

In some embodiments, the immune modulating agent inhibits an immunecheckpoint pathway. The immune system has multiple inhibitory pathwaysthat are involved in maintaining self-tolerance and for modulatingimmune responses. It is known that tumors can use certainimmune-checkpoint pathways as a major mechanism of immune resistance,particularly against T cells that are specific for tumor antigens(Pardoll, 2012, Nature Reviews Cancer 12:252-264). Because many suchimmune checkpoints are initiated by ligand-receptor interactions, theycan be readily blocked by antibodies against the ligands and/or theirreceptors.

Therefore, therapy with antagonistic molecules blocking an immunecheckpoint pathway, such as small molecules, nucleic acid inhibitors(e.g., RNAi) or antibody molecules, are becoming promising avenues ofimmunotherapy for cancer and other diseases. In contrast to the majorityof anti-cancer agents, checkpoint inhibitors do not necessarily targettumor cells directly, but rather target lymphocyte receptors or theirligands in order to enhance the endogenous antitumor activity of theimmune system. (Pardoll, 2012, Nature Reviews Cancer 12:252-264).

As used herein, the term “immune checkpoint inhibitor” refers tomolecules that totally or partially reduce, inhibit, interfere with ormodulate one or more checkpoint proteins. Checkpoint proteins regulateT-cell activation or function. These proteins are responsible forco-stimulatory or inhibitory interactions of T-cell responses. Immunecheckpoint proteins regulate and maintain self-tolerance and theduration and amplitude of physiological immune responses.

Immune checkpoint inhibitors include any agent that blocks or inhibitsin a statistically significant manner, the inhibitory pathways of theimmune system. Such inhibitors may include small molecule inhibitors ormay include antibodies, or antigen binding fragments thereof, that bindto and block or inhibit immune checkpoint receptor ligands. Illustrativeimmune checkpoint molecules that may be targeted for blocking orinhibition include, but are not limited to, PD1 (CD279), PDL1 (CD274,B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3, 4-1BB (CD137),4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, Ox40 (CD134, TNFRSF4),CXCR2, tumor associated antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9,B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules andis expressed on all NK, γδ, and memory CD8+ (αβ) T cells), CD160 (alsoreferred to as BY55) and CGEN-15049. Immune checkpoint inhibitorsinclude antibodies, or antigen binding fragments thereof, or otherbinding proteins, that bind to and block or inhibit the activity of oneor more of PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, 4-1BB, 4-1BBL, GITR,CD40, Ox40, CXCR2, TAA, B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4,VISTA, KIR, 2B4, CD160, and CGEN-15049. Illustrative immune checkpointinhibitors include Tremelimumab (CTLA-4 blocking antibody), anti-OX40,PD-L1 monoclonal antibody (Anti-B7-H1; MEDI4736), MK-3475 (PD-1blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody),BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559(anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C(anti-PDL1 antibody) and Yervoy/ipilimumab (anti-CTLA-4 checkpointinhibitor).

Programmed cell death 1 (PD1) is an immune checkpoint protein that isexpressed in B cells, NK cells, and T cells (Shinohara et al., 1995,Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, NatureReviews Cancer 12:252-264). The major role of PD1 is to limit theactivity of T cells in peripheral tissues during inflammation inresponse to infection, as well as to limit autoimmunity (Pardoll, 2012,Nature Reviews Cancer 12:252-264). PD1 expression is induced inactivated T cells and binding of PD1 to one of its endogenous ligandsacts to inhibit T-cell activation by inhibiting stimulatory kinases(Pardoll, 2012, Nature Reviews Cancer 12:252-264). PD1 also acts toinhibit the TCR “stop signal” (Pardoll, 2012, Nature Reviews Cancer12:252-264). PD1 is highly expressed on Treg cells and may increasetheir proliferation in the presence of ligand (Pardoll, 2012, NatureReviews Cancer 12:252-264). Anti-PD 1 antibodies have been used fortreatment of melanoma, non-small-cell lung cancer, bladder cancer,prostate cancer, colorectal cancer, head and neck cancer,triple-negative breast cancer, leukemia, lymphoma and renal cell cancer(Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013,Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96;Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). Exemplary anti-PD1antibodies include nivolumab (Opdivo by BMS), pembrolizumab (Keytruda byMerck), pidilizumab (CT-011 by Cure Tech), lambrolizumab (MK-3475 byMerck), and AMP-224 (Merck).

PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 andB7-DC) are ligands for PD1, found on activated T cells, B cells, myeloidcells, macrophages, and some types of tumor cells. Anti-tumor therapieshave focused on anti-PD-L1 antibodies. The complex of PD1 and PD-L1inhibits proliferation of CD8+ T cells and reduces the immune response(Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012,N Eng J Med 366:2455-65). Anti-PD-L1 antibodies have been used fortreatment of non-small cell lung cancer, melanoma, colorectal cancer,renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer,breast cancer, and hematologic malignancies (Brahmer et al., N Eng J Med366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi etal., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv MedOncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51).Exemplary anti-PD-L1 antibodies include MDX-1105 (Medarex), MEDI4736(Medimmune) MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb) andMSB0010718C.

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152,is a co-inhibitory molecule that functions to regulate T-cellactivation. CTLA-4 is a member of the immunoglobulin superfamily that isexpressed exclusively on T-cells. CTLA-4 acts to inhibit T-cellactivation and is reported to inhibit helper T-cell activity and enhanceregulatory T-cell immunosuppressive activity (Pardoll, 2012, NatureReviews Cancer 12:252-264). Although the precise mechanism of action ofCTLA-4 remains under investigation, it has been suggested that itinhibits T cell activation by outcompeting CD28 in binding to CD80 andCD86, as well as actively delivering inhibitor signals to the T cell(Pardoll, 2012, Nature Reviews Cancer 12:252-264). Anti-CTLA-4antibodies have been used in clinical trials for the treatment ofmelanoma, prostate cancer, small cell lung cancer, non-small cell lungcancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al.,2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wadaet al., 2013, J Transl Med 11:89). A significant feature of anti-CTLA-4is the kinetics of anti-tumor effect, with a lag period of up to 6months after initial treatment required for physiologic response(Pardoll, 2012, Nature Reviews Cancer 12:252-264). In some cases, tumorsmay actually increase in size after treatment initiation, before areduction is seen (Pardoll, 2012, Nature Reviews Cancer 12:252-264).Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-MyersSquibb) and tremelimumab (Pfizer). Ipilimumab has recently received FDAapproval for treatment of metastatic melanoma (Wada et al., 2013, JTransl Med 11:89). In some embodiments, the immune modulating agent isnot an anti-CTLA-4 antibody.

Lymphocyte activation gene-3 (LAG-3), also known as CD223, is anotherimmune checkpoint protein. LAG-3 has been associated with the inhibitionof lymphocyte activity and in some cases the induction of lymphocyteanergy. LAG-3 is expressed on various cells in the immune systemincluding B cells, NK cells, and dendritic cells. LAG-3 is a naturalligand for the WIC class II receptor, which is substantially expressedon melanoma-infiltrating T cells including those endowed with potentimmune-suppressive activity. An exemplary anti-LAG-3 antibodies isBMS-986016. IMP321 is a soluble version of the immune checkpointmolecule LAG-3, which activates dendritic cells, increasing antigenpresentation.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3), initiallyidentified on activated Th1 cells, has been shown to be a negativeregulator of the immune response. Blockade of TIM-3 promotes T-cellmediated anti-tumor immunity and has anti-tumor activity in a range ofmouse tumor models. Combinations of TIM-3 blockade with otherimmunotherapeutic agents such as TSR-042, anti-CD137 antibodies andothers, can be additive or synergistic in increasing anti-tumor effects.TIM-3 expression has been associated with a number of different tumortypes including melanoma, NSCLC and renal cancer, and additionally,expression of intratumoral TIM-3 has been shown to correlate with poorprognosis across a range of tumor types including NSCLC, cervical, andgastric cancers. Blockade of TIM-3 is also of interest in promotingincreased immunity to a number of chronic viral diseases. TIM-3 has alsobeen shown to interact with a number of ligands including galectin-9,phosphatidylserine and HMGB1, although which of these, if any, arerelevant in regulation of anti-tumor responses is not clear at present.

4-1BB, also known as CD137, is transmembrane glycoprotein belonging tothe TNFR superfamily. 4-1BB receptors are present on activated T cellsand B cells and monocytes. An exemplary anti-4-1BB antibody is urelumab(BMS-663513), which has potential immunostimulatory and antineoplasticactivities.

Glucocorticoid-induced TNFR family related gene (GITR) is also a memberof the TNFR superfamily. GITR is upregulated on activated T cells, whichenhances the immune system. An exemplary anti-GITR antibody is TRX518.

Cluster of differentiation 40 (CD40) is also a member of the TNFRsuperfamily. CD40 is a costimulatory protein found on antigen-presentingcells and mediates a broad variety of immune and inflammatory responses.CD40 is also expressed on some malignancies, where it promotesproliferation. Exemplary anti-CD40 antibodies are dacetuzumab (SGN-40),lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics), andCP-870,893.

Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), alsoknown as OX40 and CD134, is another member of the TNFR superfamily. OX40is not constitutively expressed on resting naïve T cells and acts as asecondary co-stimulatory immune checkpoint molecule. Exemplary anti-OX40antibodies are MEDI6469 and MOXR0916 (RG7888, Genentech).

In some embodiments, the immune modulating agent is an antibody orantigen-binding antibody fragment thereof. Exemplary of such antibodiesinclude, but are not limited to, Daclizumab (Zenapax), Bevacizumab(Avastin®), Basiliximab, Ipilimumab, Nivolumab, pembrolizumab,MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A(Atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab,PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab(HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916,AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7,Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab(BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab, CC-90002 andMNRP1685A or an antibody-binding fragment thereof.

CXCR2 is a chemokine receptor that is expressed on myeloid-derivedsuppressor cells (MDSCs). CXCR2s contribute to tumor immune escape. Ithas been shown that anti-CXCR2 monoclonal antibody therapy, enhanced ananti-PD1 antibody-induced anti-tumor immune response and anti-tumorefficacy.

In some embodiments, the immune-modulating agent is cytokine. In someembodiments, the immune modulating agent is a cytokine or is an agentthat induces increased expression of a cytokine in the tumormicroenvironment. By “cytokine” is meant a generic term for proteinsreleased by one cell population that act on another cell asintercellular mediators. Examples of such cytokines are lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormones such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-alpha and -beta; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-beta; platelet-growth factor; transforming growth factors (TGFs)such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines. For example, the immune modulating agent is acytokine and the cytokine is IL-4, TNF-α, GM-CSF or IL-2.

In some embodiments, the immune modulating agent is selected from amongGM-CSF, CpG-ODN (CpG oligodeoxynucleotides), lipopolysaccharide (LPS),monophosphoryl lipid A (MPL), alum, recombinant Leishmania polyprotein,imiquimod, MF59, poly I:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys,complete freund's adjuvant (CFA), alpha-galactosylceramide, RC-529,MDF2β, Loxoribine, anti-CD40 agonist, SIRPa antagonist, AS04, AS03,Flagellin, Resiquimod, DAP (diaminopimelic acid), MDP (muramyldipeptide) and CAF01 (cationic adjuvant formulation-01). In someembodiments, the immune modulating agent is a Toll-like receptor (TLR)agonist, an adjuvant or a cytokine. In some embodiments, the immunemodulating agent is a TLR agonist and the TLR agonist is TLR agonist isa TLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. Insome embodiments, the TLR agonist is selected from among triacylatedlipoprotein, diacylated lipopeptide, lipoteichoic acid, peptidoglycan,zymosan, Pam3CSK4, dsRNA, polyI:C, Poly G10, Poly G3, CpG, 3M003,flagellin, lipopolysaccharide (LPS) Leishmania homolog of eukaryoticribosomal elongation and initiation factor 4a (LeIF), MEDI9197, SD-101,and imidazoquinoline TLR agonists.

In some embodiments, the immune modulating agent can contain one or moreinterleukins or other cytokines. For example, the interleukin caninclude leukocyte interleukin injection (Multikine), which is acombination of natural cytokines.

In some embodiments, the immune modulating agent is a Toll-like receptor(TLR) agonist. In some embodiments, such agonists can include a TLR4agonist, a TLR8 agonist, or a TLR9 agonist. Such an agonist may beselected from peptidoglycan, polyI:C, CpG, 3M003, flagellin, andLeishmania homolog of eukaryotic ribosomal elongation and initiationfactor 4a (LeIF).

In some embodiments, the immune modulating agent can be one thatenhances the immunogenicity of tumor cells such as patupilone(epothilone B), epidermal-growth factor receptor (EGFR)-targetingmonoclonal antibody 7A7.27, histone deacetylase inhibitors (e.g.,vorinostat, romidepsin, panobinostat, belinostat, and entinostat), then3-polyunsaturated fatty acid docosahexaenoic acid, proteasomeinhibitors (e.g., bortezomib), shikonin (the major constituent of theroot of Lithospermum erythrorhizon,) and oncolytic viruses, such as TVec(talimogene laherparepvec). In some embodiments, the immune modulatingagent activates immunogenic cell death of the cancer or tumor, such asantrhacyclins (doxorubicin, mitoxantron), BK channel agonists,bortezomib, botrtezomib plus mitocycin C plus hTert-Ad, Cardiacglycosides plus non-ICD inducers, cyclophosphamide, GADD34/PP1inhibitors plus mitomycin, LV-tSMAC, and oxaliplatin. In someembodiments, the immune modulating agent can be an epigenetic therapy,such as DNA methyltransferase inhibitors (e.g., Decitabine,5-aza-2′-deoxycytidine).

For example, in some embodiments, the immune modulating agent can be aDNA methyltransferase inhibitor, which can regulate expression of tumorassociated antigens (TAA). TAAs are antigenic substances produced intumor cells which triggers an immune response. TAAs are oftendown-regulated by DNA methylation in tumors to escape the immune system.Reversal of DNA methylation restores TAA expression, increasing theimmunogencity of tumor cells. For example, demethylating agents such asdecitabine (5-aza-2′-deoxycytidine) can upregulate expression of TAAs intumor cells and increase immune recognition of the cancerous cells.Photoimmunotherapy would further expose TAAs to the immune system bydisrupting cells.

In some embodiments, the immune modulating agent itself can be anantibody conjugate containing a phthalocyanine dye linked to an antibodyor antigen-binding antibody fragment that is an immune modulating agent,such as an immune checkpoint inhibitor. In some embodiments, the immunemodulating agent is one that targets or binds to an immunosuppressivemolecule, such as an immune checkpoint molecule, on the surface of tumorcells. For example, PD-L1 is an immunosuppressive molecule that isconstitutively expressed or induced on many tumor cells, and can preventT cell activation through interactions with its receptor PD-1 expressedon immune cells. In some aspects, a phthalocyanine-dye conjugatecontaining an immune modulating agent that binds to an immunosuppressivemolecule on a tumor cells (e.g., PD-L1) can be administered both toenhance an immune response and also to specifically kill cancer cellsthat express the immunosuppressive molecule, thereby reversing immunesuppression in the tumor microenvironment. In particular, irradiation oftumor cells cells to which the conjugate binds can result in itsactivation to mediate PIT-induced cell killing of the PD-L1 cancercells, which also would act to specifically eliminate the cancer cellsin the tumor that control T-cell suppression in the tumormicroenvironment.

Hence, provided herein is a conjugate containing a phthalocyanine dye(e.g., IR700) linked to an immune modulating agent that binds to animmunosuppressive molecule expressed on tumor cells. For example, insome embodiments, the immunosuppressive molecule expressed on tumorcells can be an immune checkpoint molecule. In some embodiments, theimmune checkpoint molecule expressed on tumor cells is PD-L1. In someembodiments, the immune modulating agent that is part of the conjugateis an immune checkpoint inhibitor, such as an antibody orantigen-binding antibody fragment that binds to PD-L1. For example,provided herein is a conjugate containing a phthalocyanine dye (e.g.,IR700) linked to an antibody or antigen-binding antibody fragment thatbinds to PD-L1. Exemplary immune checkpoint inhibitors, includingantibodies or antigen-binding antibody fragments, against PD-L1 aredescribed above, and any can be included in the provided conjugates.Exemplary anti-PD-L1 antibodies include, but are not limited to,BMS-935559, MEDI4736, MPDL3280A and MSB0010718C, or an antigen-bindingantibody fragment thereof. Exemplary conjugate molecules provided hereininclude, for example, IR700-BMS-935559, IR700-MEDI4736, IR700-MPDL3280Aand IR700-MSB0010718C. In some embodiments, such conjugates can be usedin methods of photoimmunotherapy, for example, by irradiation with lightat a wavelength sufficient to activate the dye. Such conjugates can beused in monotherapy-based photoimmunotherapy or can be used incombination therapy methods with other phthalocyanine dye conjugates.

For example, in some embodiments, combination therapy methods areprovided in which a first conjugate containing a phthalocyanine dye(e.g., IR700) linked to an immune modulating agent that binds to animmunosuppressive molecule expressed on cells of a tumor (e.g., ananti-PD-L1 antibody, such as an IR700-anti-PD-L1 conjugate) isadministered to a subject, and then a second conjugate containing aphthalocyanine dye linked to a targeting molecule is administered to thesubject. Generally, the second conjugate can include any targetingmolecule that is able to bind to a cell surface protein on a cell in atumor, such as a cell present in a tumor microenvironment, such as anydescribed above. In some embodiments, the first conjugate and the secondconjugate bind to different proteins expressed on a cell in a tumor. Insome embodiments, the second conjugate can include a phthalocyanine dye(e.g., IR700) linked to an antibody or antigen-binding antibody fragmentthat binds to a cell surface protein expressed on a cell in a tumor.Exemplary antibody or antigen-binding antibody fragments of the secondconjugate can include, but are not limited to, bevacizumab, cetuximab,panitumumab, zalutumumab, nimotuzumab, Tositumomab (Bexxar®), Rituximab(Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin), Daclizumab(Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fab fragment,OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab (Avastin®), andBasiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559,MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525,urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab,SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916,AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab(CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201,AGX-115, Emactuzumab, CC-90002 and MNRP1685A or is an antibody-bindingfragment thereof.

In some embodiments, for example, if the treatment of the tumor with theconjugate followed by light irradiation increases the presence ofimmunosuppressive cells in the tumor or increases the expression ofimmunosuppressive markers at the tumor, a therapeutically effectiveamount of an immune modulating agent capable of reducing the amount oractivity of immunosuppressive cells in the tumor or capable of blockingthe activity of the immunosuppressive marker or reducing the activity ofa tumor promoting cell in the tumor or capable of blocking the activityof the tumor promoting marker can be administered. For example, in someembodiments, a conjugate with a first dye that is a phthalocyanine dyeis administered, in combination with an immune modulating agent includesa conjugate that includes a second phthalocyanine dye conjugated to animmune modulating agent capable of binding to the immunosuppressive cellor a tumor promoting cell, and modulating the activity of such cell. Insome embodiments, the first and second phthalocyanine dye is the same ordifferent.

Thus, in some embodiments, the immune modulating agent is itself aconjugate containing a phthalocyanine dye, such as a phthalocyanine dyelinked to an antibody or antigen-binding antibody fragment that is animmune modulating agent. In some embodiments, the immune modulatingagent is an IR700-antibody conjugate that includes an immune modulatingantibody (e.g., checkpoint inhibitor) that binds to a checkpoint proteinon a tumor cell (e.g., PD-L1). In some embodiments, the immunemodulating conjugate (e.g., IR700-antibody conjugate that is an immunemodulating agent) is administered prior to administration of thephthalocyanine dye-targeting molecule conjugate, such as between 12hours and 2 months, such as generally at least 12 hours, at least 24hours, at least 48 hours, at least 96 hours, at least one week, at leasttwo weeks, at least three weeks or at least one month prior toadministration of the phthalocyanine dye-targeting molecule conjugate.In some embodiments, the immune modulating conjugate (e.g.,IR700-antibody conjugate that is an immune modulating agent) isadministered during or simultaneously with administration of thephthalocyanine dye-targeting molecule conjugate. In some embodiments,the immune modulating conjugate (e.g., IR700-antibody conjugate that isan immune modulating agent) is administered after administration of thephthalocyanine dye-targeting molecule conjugate, such as between 12hours and 2 months, such as generally at least 12 hours, at least 24hours, at least 48 hours, at least 96 hours, at least one week, at leasttwo weeks, at least three weeks or at least one month afteradministration of the phthalocyanine dye-targeting molecule conjugate,or the irradiation after administration of the phthalocyaninedye-targeting molecule conjugate.

In such aspects, the combination therapy methods generally include oneor more irradiations with light at a wavelength sufficient to activatethe dye of the first and/or second conjugate.

In some embodiments, at least two irradiations are performed, where atleast a first irradiation is provided to activate the first conjugateand a second irradiation is provided to activate the second conjugate.In some embodiments, a first irradiation with light is provided to thetumor after administration of the first conjugate. For example, from orfrom about 12 hours to 48 hours, such as about or approximately within24 hours, after administering the first conjugate, the tumor can betreated with light to kill cancer cells that express theimmunosuppressive molecule, such as to kill tumor cells that expressPD-L1. In some embodiments, the killing of such cells may permitre-activation of or amplification of T cell responses at the tumor. Insome embodiments, subsequent to photoimmunotherapy of the firstconjugate by administration and irradiation, the second phthalocyaninedye conjugate can be administered to the subject, followed by a secondirradiation with light from or from about 12 hours to 48 hours, such asabout or approximately within 24 hours, after administering the secondconjugate. In some embodiments, the second irradiation achievesactivation of the second conjugate, which can result in selective cellkilling of tumor cells that express the tumor-targeted moleculerecognized by the second conjugate, thereby releasing tumor antigens toinduce a strong immunogenic response as the T cell in the tumor are nolonger suppressed by the immunosuppressive molecule (e.g., PD-L1). Insome embodiments, the first irradiation is performed prior toadministration of the second conjugate, such as at least or about atleast 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 12 hours or 24 hours prior to administration of thesecond conjugate.

In some embodiments, a single irradiation can performed to effectactivation of both the first conjugate and the second conjugate in ordercause PIT-induced cell killing of tumor cells expressing theimmunosuppressive molecule (e.g., PD-L1) recognized by the firstconjugate and tumor cells expressing the tumor-targeted moleculerecognized by the second conjugate. Hence, in such aspects, the onelight irradiation of the tumor may induce both effects to selectivelykill specific tumor cells, thereby releasing tumor antigens, as well asinducing a strong immunogenic response due to the killing of theimmunosuppressive tumor cells, such as the tumor cells expressing PD-L1.In some embodiments, prior to the irradiation, the first conjugate canbe administered prior, simultaneously, subsequently or intermittentlyfrom administration of the second conjugate. In some embodiments, thefirst conjugate is administered prior to the second conjugate, such asat least 5 minutes prior, and generally at least 12 hours or at least 24hours prior. In some embodiments, the first and second conjugates areadministered simultaneously. In some embodiments, the first and secondconjugates are formulated separately. In some embodiments, the first andsecond conjugates are formulated together in the same composition.

2. Anti-Cancer Agents

Also provided herein are anti-cancer agents that can be administered incombination with photoimmunotherapy employing phthalocyaninedye-targeting molecule conjugates. Hence, the combination therapyprovided herein, including combinations and methods of use thereof,include an anti-cancer agent, which can include any agent whose use canreduce, arrest or prevent cancer in a subject. Optionally, an additionalanti-cancer agent can be used in combination therapy withphotoimmunotherapy using phthalocyanine dye-targeting moleculeconjugates together with an immune modulating agent, for example totreat various cancers.

As described herein, PIT-induced cell killing of tumor cells byadministration of one or more phthalocyanine dye conjugates to a subjecthaving a tumor in combination with irradiation can lead to increases intumor permeability, such as increases in vascular permeability aroundthe tumor space. It is believed herein that the increase in permeabilitycan result in rapid leakage of systemically available molecules into thetumor space, thereby maximizing exposure of the tumor to such molecules.Thus, in some embodiments, in the combination therapy methods providedherein, an anti-cancer agent is administered to a subject a sufficienttime prior to irradiation of an administered phthalocyaninedye-targeting molecule conjugate to render the anti-cancer agentsystemically available, such as generally at least 5 minutes prior toirradiation, for example at least 10 minutes, 15 minutes, 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 24 hoursprior to irradiation. In such embodiments, following irradiation andPIT-induced killing of tumor cells, the systemically availableanti-cancer agent can be immediately taken up into the tumor space wherethe agent can provide a therapeutic effect. Thus, in contrast to methodsin which the anti-cancer agent is administered after irradiation, andhence after PIT-induced cell killing, in the instant methods there is nolag time in achieving a therapeutic effect because the anti-cancer agentis available for direct and immediate uptake into the tumor space. Thiscan maximize therapeutic responses to the anti-cancer agent.

It is within the level of a skilled artisan to determine the appropriatetiming of administration of a particular anti-cancer agent prior toperforming irradiation to ensure sufficient systemic availability of theanti-cancer agent. In many cases, the pharmacokinetics of particularanti-cancer agents are well known in the art. In some cases,pharmacokinetics can be assessed by measuring such parameters as themaximum (peak) plasma concentration (C_(max)), the peak time (i.e. whenmaximum plasma concentration occurs; T_(max)), the minimum plasmaconcentration (i.e. the minimum plasma concentration between doses ofagent; C_(min)), the elimination half-life (T_(1/2)) and area under thecurve (i.e. the area under the curve generated by plotting time versusplasma concentration of the agent; AUC), following administration. Theconcentration of a particular agent in the plasma following subcutaneousadministration can be measured using any method known in the artsuitable for assessing concentrations of agents in samples of blood. Forexample, an immunoassay, such as an ELISA, or chromatography/massspectrometry-based assays can be used.

In some embodiments, the anti-cancer agent that is used in thecombination therapy provided herein can refer to any agents, orcompounds, used in anti-cancer treatment. These include any agents, whenused alone or in combination with other compounds, that can alleviate,reduce, ameliorate, prevent, or place or maintain in a state ofremission of clinical symptoms or diagnostic markers associated withtumors and cancer, and can be used in combinations and compositionsprovided herein. In some embodiments, the anti-cancer agent is one whosetherapeutic effect is generally associated with penetration or deliveryof the anti-cancer agent into the tumor microenvironment or tumor space.In some embodiments, the anti-cancer agent is an alkylating agent, aplatinum drug, an antimetabolite, an anti-tumor antibiotic, atopoisomerase inhibitor, a mitotic inhibitor, a corticosteroid, aproteasome inhibitor, a kinase inhibitor, a histone-deacetylaseinhibitor or an antibody or antigen-binding antibody fragment thereof.In some embodiments, the anti-cancer agent is a peptide, protein orsmall molecule drug.

In some embodiments, the anti-cancer agent is 5-Fluorouracil/leukovorin,oxaliplatin, irinotecan, regorafenib, ziv-afibercept, capecitabine,cisplatin, paclitaxel, toptecan, carboplatin, gemcitabine, docetaxel,5-FU, ifosfamide, mitomycin, pemetrexed, vinorelbine, carmustine wager,temozolomide, methotrexate, capacitabine, lapatinib, etoposide,dabrafenib, vemurafenib, liposomal cytarabine, cytarabine, interferonalpha, erlotinib, vincristine, cyclophosphamide, lomusine, procarbazine,sunitinib, somastostatin, doxorubicin, pegylated liposomal encapsulateddoxorubicin, epirubicin, eribulin, albumin-bound paclitaxel,ixabepilone, cotrimoxazole, taxane, vinblastine, temsirolimus,temozolomide, bendamustine, oral etoposide, everolimus, octreotide,lanredtide, dacarbazine, mesna, pazopanib, eribulin, imatinib,regorafenib, sorafenib, nilotinib, dasantinib, celecoxib, tamoxifen,toremifene, dactinomycin, sirolimus, crizotinib, certinib, enzalutamide,abiraterone acetate, mitoxantrone, cabazitaxel, fluoropyrimidine,oxaliplatin, leucovorin, afatinib, ceritinib, gefitinib, cabozantinib,oxoliplatin or auroropyrimidine.

In some embodiments, the anti-cancer agent is an antibody orantigen-binding antibody fragment. In some embodiments, the anti-canceragent can be any one or more of bevacizumab, cetuximab, panitumumab,ramucirumab, ipilimumab, rituximab, trastuzumab, ado-trastuzumabemtansine, pertuzumab, nivolumab, lapatinib, dabrafenib, vemurafenib,erlotinib, sunitinib, pazopanib, imatinib, regorafenib, sorafenib,nilotinib, dasantinib, celecoxib, crizotinib, certinib, afatinib,axitinib, bevacizumab, bosutinib, cabozantinib, afatinib, gefitinib,temsirolimus, everolimus, sirolimus, ibrutinib, imatinib, lenvatinib,olaparib, palbociclib, ruxolitinib, trametinib, vandetanib orvismodegib, or an antigen-binding antibody fragment thereof.

In some embodiments, the anti-cancer agent is an alkylating agent.Alkylating agents are compounds that directly damage DNA by formingcovalent bonds with nucleic acids and inhibiting DNA synthesis.Exemplary alkylating agents include, but are not limited to,mechlorethamine, cyclophosphamide, ifosamide, melphalan, chlorambucil,busulfan, and thiotepa as well as nitrosurea alkylating agents such ascarmustine and lomustine.

In some embodiments, the anti-cancer agent is a platinum drug. Platinumdrugs bind to and cause crosslinking of DNA, which ultimately triggersapoptosis. Exemplary platinum drugs include, but are not limited to,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,nedaplatin, triplatin, and lipoplatin.

In some embodiments, the anti-cancer agent is an antimetabolite.Antimetabolites interfere with DNA and RNA growth by substituting forthe normal building blocks of RNA and DNA. These agents damage cellsduring the S phase, when the cell's chromosomes are being copied. Insome cases, antimetabolites can be used to treat leukemias, cancers ofthe breast, ovary, and the intestinal tract, as well as other types ofcancer. Exemplary antimetabolites include, but are not limited to,5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®),cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®),hydroxyurea, methotrexate, and pemetrexed (Alimta®).

In some embodiments, the anti-cancer agent is an anti-tumor antibiotic.Anti-tumor antibiotics work by altering the DNA inside cancer cells tokeep them from growing and multiplying. Anthracyclines are anti-tumorantibiotics that interfere with enzymes involved in DNA replication.These drugs generally work in all phases of the cell cycle. They can bewidely used for a variety of cancers. Exemplary anthracyclines include,but are not limited to, daunorubicin, doxorubicin, epirubicin, andidarubicin. Other anti-tumor antibiotics include actinomycin-D,bleomycin, mitomycin-C, and mitoxantrone.

In some embodiments, the anti-cancer agent is a topoisomerase inhibitor.These drugs interfere with enzymes called topoisomerases, which helpseparate the strands of DNA so they can be copied during the S phase.Topoisomerase inhibitors can be used to treat certain leukemias, as wellas lung, ovarian, gastrointestinal, and other cancers. Exemplarytoposiomerase inhibitors include, but are not limited to, doxorubicin,topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, andmitoxantrone.

In some embodiments, the anti-cancer agent is a mitotic inhibitor.Mitotic inhibitors are often plant alkaloids and other compounds derivedfrom natural plant products. They work by stopping mitosis in the Mphase of the cell cycle but, in some cases, can damage cells in allphases by keeping enzymes from making proteins needed for cellreproduction. Exemplary mitotic inhibitors include, but are not limitedto, paclitaxel (Taxol®), docetaxel (Taxotere®), ixabepilone (Ixempra®),vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®),and estramustine (Emcyt®).

In some embodiments, the anti-cancer agent is a corticosteroid.Corticosteroids, often simply called steroids, are natural hormones andhormone-like drugs that are useful in the treatment of many types ofcancer. Corticosteroids can also be used before chemotherapy to helpprevent allergic reactions as well as during and after chemotherapy tohelp prevent nausea and vomiting. Exemplary corticosteroids include, butare not limited to, prednisone, methylprednisolone (Solumedrol®), anddexamethasone (Decadron®).

In some embodiments, the anti-cancer agent is another type ofchemotherapy drug, such as a proteosome inhibitor, a kinase inhibitor,or a histone-deacetylase inhibitor. In other embodiments, theanti-cancer agent is a biologic such as an antibody used in cancertherapy.

In some embodiments, the anti-cancer agent targets tumors associatedwith various cancers. The cancer can be any cancer located in the bodyof a subject, such as, but not limited to, cancers located at the headand neck, breast, liver, colon, ovary, prostate, pancreas, brain,cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, orlung. For example, the anti-cancer agent can be used for the treatmentof colon cancer, cervical cancer, cancer of the central nervous system,breast cancer, bladder cancer, anal carcinoma, head and neck cancer,ovarian cancer, endometrial cancer, small cell lung cancer, non-smallcell lung carcinoma, neuroendocrine cancer, soft tissue carcinoma,penile cancer, prostate cancer, pancreatic cancer, gastric cancer, gallbladder cancer or esophageal cancer. In some cases, the cancer can be acancer of the blood.

E. Exemplary Features

In some embodiments, a desired response of treatment according to theprovided methods is to reduce or inhibit one or more symptoms associatedwith a tumor or a cancer. In some embodiments, the one or more symptomsdo not have to be completely eliminated for the composition to beeffective.

For example, administration of a composition containing thephthalocyanine dye-targeting molecule conjugate followed by irradiationcan decrease the size of a tumor, such as the volume or weight of atumor, or metastasis of a tumor, for example by at least 20%, at least305, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, or at least 100%, ascompared to the tumor size, volume, weight, or metastasis in the absenceof the conjugate. In some embodiments, the difference in tumor size,volume, weight or metastasis is evident after at least 7 days, at least10 days, at least 14 days, at least 30 days, at least 60 days, at least90 days, or at least 120 days after the treatment(s). In someembodiments, tumor size and volume can be monitored by radiography,ultrasound imaging, necropsy, by use of calipers, by microCT or by¹⁸F-FDG-PET. Tumor size also can be assessed visually. In particularexamples, tumor size (diameter) can be measured directly using calipers.

In some embodiments, combining the phthalocyanine dye-targeting moleculeconjugates and PIT (e.g. antibody-IR700 molecules/PIT) with theadditional therapy, such as an immune modulating agent or anti-canceragent, in accord with the methods herein can result in a tumor size,volume, weight or metastasis that is less than the tumor size, volume,weight or metastasis would be if it were treated with either thephthalocyanine dye-targeting molecule conjugate/PIT alone or theadditional therapy alone, that is, there is a synergistic effect. Forexample, the combination therapy provided herein can decrease the sizeof a tumor, such as the volume or weight of a tumor, or metastasis of atumor, for example by at least 1.2-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more ascompared to the tumor size, volume, weight, or metastasis achieved intherapy methods involving only photoimmunotherapy with a compositioncontaining the phthalocyanine dye-targeting molecule conjugate followedby irradiation or in therapy methods involving monotherapy with theimmune modulating agent or anti-cancer agent alone.

In some embodiments, a desired response of treatment according to theprovided methods is to kill a population of cells by a desired amount,for example by killing at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or atleast 100% of the cells, as compared to cell killing in the absence ofthe conjugate and irradiation. In some embodiments, the difference intumor cell killing is evident after at least 1 hour, at least 2 hours,at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, at least 10 days, at least 14 days or at least 30 days,after the treatment(s). In some embodiments, cell killing activity canbe assessed by a variety of techniques known in the art including, butnot limited to, cytotoxicity/cell viability assays that can be employedto measure cell necrosis and/or apoptosis, such as from a biopsy sample,following treatment(s), such as MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assaysand other related tetrazolium salt based assays (e.g., XTT, MTS or WST),ATP assays, apoptosis assays (e.g., using labeled annexin V), such asTUNEL staining of infected cells, DNA fragmentation assays, DNAladdering assays, and cytochrome C release assays. In some cases,imaging methods can be used, such as positron emission tomography (PET),including FDG-PET, single photon emission CT (SPECT), diffusion weightedimaging (DWI), dynamic susceptibility-weighted contrast-enhanced (DSC)MR imaging or dynamic contrast-enhanced (DCE) MR imaging, CT perfusionmethods, magnetic resonance spectroscopy (MRS) Such assays and methodsare well known to one of skill in the art.

In some embodiments, the combination therapy provided herein canincrease the killing of tumor cells, for example, by at least by atleast 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or more as compared to cell killing intherapy methods involving only photoimmunotherapy with a compositioncontaining the phthalocyanine dye-targeting molecule conjugate followedby irradiation or in therapy methods involving monotherapy with theimmune modulating agent or anti-cancer agent alone.

In some embodiments, a desired response is to increase the survival timeof a patient with a tumor, or who has had a tumor recently removed, by adesired amount, for example to increase survival by at least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or at least 100%, as compared to the survivaltime in the absence of the conjugate and irradiation. In someembodiments, increased survival is evident by an increase in one or moresurvival indicators from among duration of median progression-freesurvival, duration of response, median overall survival or othersurvival-related clinical endpoint. In some embodiments, the differencein survival is evident after at least 7 days, at least 10 days, at least14 days, at least 30 days, at least 60 days, at least 90 days, at least120 days, at least 6 months, at least 12 months, at least 24 months, orat least 5 years or more after the treatment(s). In some embodiments,antibody-IR700 molecules/PIT alone in accord with the methods herein,increases the duration of median progression-free survival, duration ofresponse, median overall survival or other survival-related clinicalendpoint by at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months, at least 12 months, at least 18months, at least 24 months, or at least 5 years or more compared to if asubject were treated with the corresponding targeting molecule that wasnot so conjugated. In some embodiments, antibody-IR700 molecules/PIT incombination the additional therapy, such as an immune modulating agentor anti-cancer agent, in accord with the methods herein, increases theduration of median progression-free survival, duration of response,median overall survival or other survival-related clinical endpoint byat least 3 months, at least 4 months, at least 5 months, at least 6months, at least 7 months, at least 8 months, at least 9 months, atleast 10 months, at least 11 months, at least 12 months, at least 18months, at least 24 months, or at least 5 years or more compared to ifthe subject were treated with the phthalocyanine dye-targeting moleculeconjugate/PIT alone or the additional therapy alone.

In some embodiments, the combination therapy provided herein canincrease the survival time of a treated subject, for example, by atleast 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or more as compared to the survival timein a subject receiving a therapy involving only photoimmunotherapy witha composition containing the phthalocyanine dye-targeting moleculeconjugate followed by irradiation or in therapy methods involvingmonotherapy with the immune modulating agent or anti-cancer agent alone.In some embodiments, combining the antibody-IR700 molecules/PIT with theadditional therapy, such as an immune modulating agent or anti-canceragent, in accord with the methods herein, increases the duration ofmedian progression-free survival, duration of response, median overallsurvival or other survival-related clinical endpoint by at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, at least 18 months, atleast 24 months, or at least 5 years or more compared to if it weretreated with either the phthalocyanine dye-targeting moleculeconjugate/PIT alone or the additional therapy alone.

In one aspect, the response to treatment is characterized utilizingResponse Evaluation Criteria in Solid Tumors (RECIST) criteria, which isthe recommended guideline for assessment of tumor response by theNational Cancer Institute (see Therasse et al., J. Natl. Cancer Inst.92:205-216, 2000). In some embodiments, patients can be assessed forresponse to the therapy using RECIST criteria as outlined in the revisedversion 1.1 guidelines (RECIST 1.1, see Eisenhauer et al. (2009)European Journal of Cancer, 45:228-247). The criteria for objectivestatus are required for protocols to assess solid tumor response.Representative criteria include the following: (1) Complete Response(CR), defined as complete disappearance of all measurable disease; nonew lesions; no disease related symptoms; no evidence of non-measurabledisease; (2) Partial Response (PR) defined as 30% decrease in the sum ofthe longest diameter of target lesions (e.g., tumor); (3) ProgressiveDisease (PD), defined as 20% increase in the sum of the longest diameterof target lesions or appearance of any new lesion; (4) Stable or NoResponse, defined as not qualifying for CR, PR, or PD. (See Therasse etal., supra.) In some embodiments, the objective response rate (ORR) canbe determined, which is the percentage of subjects in which a CR or PRresponse is observed. ORR is commonly used to measure tumor response totreatment in oncology clinical trials.

In some embodiments, administration of the phthalocyanine dye-targetingmolecule conjugate in accord with the provided methods, either as amonotherapy or in a combination therapy, achieves a reduction in thesize or volume of the tumor by at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80% at least 90% or more within twoweeks or one month of the irradiation compared to the size or volume ofthe tumor prior to the administration and irradiation.

In some embodiments, in a population of treated subjects, administrationof the phthalocyanine dye-targeting molecule conjugate in accord withthe provided methods, either as a monotherapy or as a combinationtherapy, effects an improvement of a disorder- or cancer-relatedparameter compared to a similarly situated population of subjectstreated with the targeting molecule (e.g., antibody or antigen-bindingantibody fragment) that is not conjugated, wherein the parameter isselected from one or more of: a) objective response rate (ORR); b)progression free survival (PFS); c) overall survival (OS); d) reductionin toxicity; e) tumor response; of f) quality of life. In someembodiments, the parameter is improved by at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100% or more.

In some embodiments, in a population of treated subjects, administrationof the phthalocyanine dye-targeting molecule conjugate in accord withthe provided methods, either as a monotherapy or in a combinationtherapy, results in a PR in at least 50%, 60%, 70%, 80%, 90%, 95% or100% of the treated subjects. In some embodiments, in a population oftreated subjects, administration of the phthalocyanine dye-targetingmolecule conjugate in accord with the provided methods results in a CRin at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% ofthe treated subjects.

In some embodiments, in a population of treated subjects, administrationof the phthalocyanine dye-targeting molecule conjugate in accord withthe provided methods, either as a monotherapy or in a combinationtherapy, results in an ORR that is greater than about 13%, for examplegreater than about 15%, greater than about 20%, greater than about 30%,greater than about 40%, greater than about 50%, greater than about 60%,greater than about 70%, greater than about 80%, greater than about 95%,or greater than about 99%.

In some embodiments, the combination therapy provided herein, such astherapies that employing an immune modulating agent, can be used tostimulate an immune response in a cancer patient. Typically, immuneresponses may be detected by any of a variety of well-known parameters,including but not limited to in vivo or in vitro determination of:soluble immunoglobulins or antibodies; soluble mediators such ascytokines, lymphokines, chemokines, hormones, growth factors and thelike as well as other soluble small peptide, carbohydrate, nucleotideand/or lipid mediators; cellular activation state changes as determinedby altered functional or structural properties of cells of the immunesystem, for example cell proliferation, altered motility, induction ofspecialized activities such as specific gene expression or cytolyticbehavior; cellular differentiation by cells of the immune system,including altered surface antigen expression profiles or the onset ofapoptosis (programmed cell death); an increase in cytotoxic T-cells,activated macrophages or natural killer cells; or any other criterion bywhich the presence of an immune response may be detected.

Procedures for performing these and similar assays are widely known andmay be found, for example in Lefkovits (Immunology Methods Manual: TheComprehensive Sourcebook of Techniques, 1998; see also Current Protocolsin Immunology; see also, e.g., Weir, Handbook of ExperimentalImmunology, 1986 Blackwell Scientific, Boston, Mass.; Mishell and Shigii(eds.) Selected Methods in Cellular Immunology, 1979 Freeman Publishing,San Francisco, Calif.; Green and Reed, 1998 Science 281:1309 andreferences cited therein.).

Detection of the proliferation of tumor-reactive T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring the rate of DNA synthesis,and tumor specificity can be determined by controlling the stimuli (suchas, for example, a specific desired tumor- or a control antigen-pulsedantigen presenting cells) to which candidate tumor-reactive T cells areexposed. T cells which have been stimulated to proliferate exhibit anincreased rate of DNA synthesis. A typical way to measure the rate ofDNA synthesis is, for example, by pulse-labeling cultures of T cellswith tritiated thymidine, a nucleoside precursor which is incorporatedinto newly synthesized DNA. The amount of tritiated thymidineincorporated can be determined using a liquid scintillationspectrophotometer. Other ways to detect T cell proliferation includemeasuring increases in interleukin-2 (IL-2) production, Ca²⁺ flux, ordye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumAlternatively, synthesis of lymphokines (such as interferon-gamma) canbe measured or the relative number of T cells that can respond to aparticular antigen may be quantified.

Detection of antibody production (e.g., tumor specific antibodyproduction) may be achieved, for example, by assaying a sample (e.g., animmunoglobulin containing sample such as serum, plasma or blood) from ahost treated with a composition according to the present invention usingin vitro methodologies such as radioimmunoassay (MA), enzyme linkedimmunosorbent assays (ELISA), equilibrium dialysis or solid phaseimmunoblotting including Western blotting. In preferred embodimentsELISA assays may further include tumor antigen-capture immobilization ofa target tumor antigen with a solid phase monoclonal antibody specificfor the antigen, for example, to enhance the sensitivity of the assay.Elaboration of soluble mediators (e.g., cytokines, chemokines,lymphokines, prostaglandins, etc.) may also be readily determined byenzyme-linked immunosorbent assay (ELISA), for example, using methods,apparatus and reagents that are readily available from commercialsources (e.g., Sigma, St. Louis, Mo.; see also R & D Systems 2006Catalog, R & D Systems, Minneapolis, Minn.).

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5th Ed., 1997 American Society of Microbiology,Washington, D.C.

IV. Imaging

In some embodiments, administration of the conjugate to the subject alsocan facilitate imaging of the subject for fluorescence signal. In someembodiments, the conjugates can be used as in vitro or in vivo opticalimaging agents of cells, tissues or organs in various biomedicalapplications. In some embodiments, the phthalocyanine dye-targetingmolecule conjugates can be used as in vivo optical imaging agents oftumors, tissues, and organs in a subject. In some embodiments, thephthalocyanine dye-targeting molecule conjugates are used for thedetection of tumors and other abnormalities. For example, the existenceof cancer cells or cancer tissues can be verified by administering thephthalocyanine dye-targeting molecule conjugate to the subject fordetection and imaging of the tumor.

In some embodiments, the detection or evaluation of a fluorescencesignal can be used to monitor uploading of the conjugate at the lesion(e.g., tumor) prior to PIT. In some embodiments, the detection orevaluation of a fluorescence signal can be used to monitor the locationof the lesion (e.g., tumor) by illuminating the area around or near thelesion to which the conjugate is selectively localized uponadministration. In some embodiments, the detection or evaluation of afluorescence signal can be used to visualize any residual cancer cellsthat may be present in a surgical setting, for example, after tumorresection, which, in some cases, can be used to facilitate targeting ofsuch cells by PIT.

In some embodiments, the fluorescence signal of the phthalocyanine dyeused for PIT (e.g., IR700) can be directly monitored.

In some embodiments, the phthalocyanine dye-targeting molecule conjugatemay contain an additional dye as described above, which, in some cases,has an emission and excitation wavelength that is different than thephthalocyanine dye (e.g., IR700). In some embodiments, thephthalocyanine dye-targeting molecule conjugates of Formula I or FormulaII are used for imaging of a subject. In some embodiments, thephthalocyanine dye-targeting molecule conjugates are administered to asubject, and irradiation or illumination is performed to identify,detect, locate, and/or follow the movement of the conjugate in thesubject. In some embodiments, imaging of the subject is performed byilluminating at a wavelength capable of being absorbed by the second oraddition dye of the conjugate but not by the phthalocyanine dye.

In some embodiments, light at a wavelength corresponding to that whichis absorbed by the dye is exposed to the conjugate. In some embodiments,the targeted area to which the phthalocyanine dye-targeting moleculeconjugates bind is exposed to light of the wavelength of electromagneticradiation absorbed by the dye, such as the phthalocyanine dye (e.g.,IR700) or the additional or second dye. In some embodiments, theconjugates, when exposed to light of an appropriate wavelength, absorbthe light, causing substances to be produced that illuminate the targetcells or tissue within the subject to which the conjugate is bound. Insome embodiments, illumination is performed to identify, detect, locate,and/or characterize a cancer cell or tumor in the subject.

In some embodiments, the conjugate is exposed to light using a deviceselected from among a hand-held ultraviolet lamp, a mercury lamp, axenon lamp, a laser, a laser diode or an LED imaging device. In someembodiments, the LED imaging device contains a near-infrared (NIR) LED.

In some embodiments, irradiation is carried out using a microlens-tippedfiber for surface illumination. In some embodiments, irradiation iscarried out using cylindrical diffusing fibers. In some embodiments, thecylindrical diffusing fibers have a diffuser length of 0.5 cm to 10 cmand are spaced 1.8±0.2 cm apart. In some embodiments, the cylindricaldiffusing fibers are placed in a catheter positioned in the tumor1.8±0.2 cm apart. In some embodiments, the catheter is opticallytransparent.

In some embodiments, the phthalocyanine dye-targeting moleculeconjugates of Formula I or Formula II are used for fluorescence imagingin surgery. In some embodiments, the phthalocyanine dye-targetingmolecule conjugates are used to image the targeted area (e.g., tumor)prior to surgery to provide information about tumor location. In someembodiments, the phthalocyanine dye-targeting molecule conjugates areused to image the targeted area at and around the tumor so that themargins of the tumor can be visualized with fluorescence and residualcancer cells in the margins can be eradicated with PIT. In someembodiments, presurgery imaging methods include but are not limited tomagnetic resonance imaging (MM) and computerized tomography (CT).

In some embodiments, the conjugates can be used to directly stain orlabel a sample so that the sample can be identified or quantified. Forinstance, the conjugate can be added as part of an assay for abiological target analyte, or as a detectable tracer element in abiological or non-biological fluid. Typically, the sample is obtaineddirectly from a liquid source or as a wash from a solid material or agrowth medium in which cells have been introduced for culturing, or abuffer solution in which cells have been placed for evaluation. Wherethe sample comprises cells, the cells are optionally single cells,including microorganisms, or multiple cells associated with other cellsin two or three dimensional layers, including multicellular organisms,embryos, tissues, biopsies, filaments, biofilms, and the like. In someembodiments, imaging of the cells or tissues is performed by irradiatingor illuminating at a wavelength capable of being absorbed by one or moreof the dyes of the conjugate. In some embodiments, in vitro imagingmethods include but are not limited to phase contrast microscopy,fluorescent microscopy, multiphoton microscopy, confocal laser scanningmicroscopy, confocal Raman microscopy, magnetic resonance microscopy,optical coherence tomography, and electron microscopy.

V. Definitions

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.” It is understood thataspects and variations described herein include “consisting” and/or“consisting essentially of” aspects and variations.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, a “conjugate” refers to a polypeptide linked directly orindirectly to one or more other polypeptides or chemical moieties. Suchconjugates include fusion proteins, those produced by chemicalconjugates and those produced by any other methods. For example, aconjugate can refer to a phthalocyanine dye, such as an IR700 molecule,linked directly or indirectly to one or more other polypeptides orchemical moieties, such as to a targeting molecule that binds to ortargets to a cell surface protein.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a “pharmaceutical composition” or “pharmaceuticalformulation” refers to a preparation which is in such form as to permitthe biological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

As used herein, a “pharmaceutically acceptable carrier” refers to aningredient in a pharmaceutical formulation, other than an activeingredient, which is nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, a derivative refers to a form of a drug that hasundergone change or modification from a reference drug or agent, butstill retains activity (e.g., exhibits increased or decreased activity)compared to the reference drug or agent. Typically a derivative form ofa compound means that a side chain of the compound has been modified orchanged.

As used herein, an analogue or analog of a drug or agent is a drug oragent that is related to a reference drug, but whose chemical andbiological activities can be different. Typically, analogues exhibitsimilar activities to a reference drug or agent, but the activity can beincreased or decreased or otherwise improved. Typically, an analogueform of a compound or drug means that the backbone core of the structureis modified or changed compared to a reference drug.

As used herein, a kit is a packaged combination that optionally includesother elements, such as additional reagents and instructions for use ofthe combination or elements thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, an “article of manufacture” is a product that is madeand, in some cases, that can be sold. In some embodiments, the term canrefer to compositions contained in articles of packaging, such as in acontainer.

As used herein, “combination therapy” refers to a treatment in which asubject is given two or more therapeutic agents, such as at least two orat least three therapeutic agents, for treating a single disease. Insome embodiments, each therapy can result in an independentpharmaceutical effect, and together can result in an additive orsynergistic pharmaceutical effect.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treating encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, “therapeutic effect” means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect. Hence, it is thequantity necessary for preventing, curing, ameliorating, arresting orpartially arresting a symptom of a disease or disorder.

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

VI. Exemplary Embodiments

Among the provided embodiments are:

1. A method of treating a disease or condition in a subject, comprising:

a) administering to a subject having a disease or condition a conjugatecomprising a phthalocyanine dye linked to a targeting molecule thatbinds to a protein on the surface of a cell present in themicroenvironment of a lesion associated with the disease or condition,wherein the conjugate is administered to effect a systemic exposure thatis no more than 75% of the therapeutically effective systemic exposureof the antibody or antigen-binding antibody fragment that is not soconjugated for treating the same disease or condition; and

b) after administering the conjugate, irradiating the lesion at awavelength of 500 nm to 900 nm at a dose of at least 1 J cm⁻² or 1 J/cmof fiber length, thereby treating the disease in the subject.

2. The method of embodiment 1, wherein the wavelength is 600 nm to 850nm.

3. The method of embodiment 1 or embodiment 2, wherein the wavelength is660 nm to 740 nm.

4. The method of any of embodiments 1-3, wherein the conjugate isadministered in a dosing schedule in which:

the administration of the conjugate is performed only one time as asingle injection or infusion; or

the dosing schedule does not comprise a subsequent dose of theconjugate; or

the dosing schedule does not comprise a subsequent dose of themacromolecule that is not so conjugated.

5. The method of any of embodiments 1-4, wherein the conjugate isadministered systemically.

6. The method of any of embodiments 1-5, wherein the conjugate isadministered intravenously.

7. The method of any of embodiments 1-6, wherein the conjugate isadministered to effect a systemic exposure (AUC) that is no more than60%, no more than 50%, no more than 40% or no more than 30% of thetherapeutically effective systemic exposure of the antibody orantigen-binding antibody fragment that is not so conjugated for treatingthe same disease or condition.

8. The method of any of embodiments 1-7, wherein the disease orcondition is a tumor, whereby the antibody or an antigen-bindingantibody fragment binds to a molecule on the surface of a cell presentin the tumor microenvironment and the tumor is irradiated.

9. The method of any of embodiments 1-8, wherein:

the systemic exposure as measured by the average area under the plasmaconjugate concentration-time curve from time 0 to infinity (AUC[0-inf])for a patient population after administration of the conjugate isbetween or between about 250 μg/mL*h and 100,000 μg/mL*h, between orbetween about 500 μg/mL*h and 50,000 μg/mL*h, between or between about500 μs/mL*h and 18,000 μg/mL*h; between or between about 500 μg/mL*h and10,000 μs/mL*h; or

the systemic exposure as measured by the average area under the plasmaconjugate concentration-time curve from time 0 to infinity (AUC[0-inf])for a patient population after administration of the conjugate is nomore than 100,000 μg/mL*h, no more than 75,000 μg/mL*h, no more than50,000 μg/mL*h, no more than 40,000 μg/mL*h, no more than 30,000μg/mL*h, no more than 20,000 μg/mL*h, no more than 10,000 μg/mL*h, nomore than 5,000 μg/mL*h, no more than 2,500 μg/mL*h.

10. The method of any of embodiments 1-9, wherein:

the systemic exposure as measured by the average area under the plasmaconjugate concentration-time curve from time 0 to 24 hours (AUC[0-24])for a patient population after administration of the conjugate isbetween or between about 100 μg/mL*h and 25,000 μg/mL*h, between orbetween about 200 μg/mL*h and 10,000 μg/mL*h, between or between about500 μs/mL*h and 5,000 μg/mL*h; or

the systemic exposure as measured by the average area under the plasmaconjugate concentration-time curve from time 0 to 24 hours (AUC[0-24])for a patient population after administration of the conjugate is nomore than 25,000 μg/mL*h, no more than 15,000 μg/mL*h, no more than10,000 μg/mL*h, no more than 5,000 μg/mL*h, no more than 2,500 μg/mL*h,no more than 1,000 μg/mL*h, or no more than 500 μg/mL*h.

11. The method of any of embodiments 1-10, wherein the conjugate isadministered in a dosage range that is at least about 10 mg/m² (bodysurface area of the subject), at least about 50 mg/m² or at least about75 mg/m² and is no more than 5000 mg/m², no more than 2000 mg/m², nomore than 1000 mg/m², no more than 500 mg/m², no more than 250 mg/m² orno more than 200 mg/m².

12. The method of any of embodiments 1-11, wherein the conjugate isadministered at a dosage that is between or between about 100 mg/m² and1500 mg/m² or 150 mg/m² and 750 mg/m².

13. The method of any of embodiments 1-12, wherein the conjugate isadministered at a dosage that is or is about 160 mg/m², 320 mg/m², 640mg/m² or 1280 mg/m².

14. The method of any of embodiments 1-13, wherein the targetingmolecule is an antibody or an antigen-binding antibody fragment.

15. The method of embodiment 14, wherein the antibody is anantigen-binding antibody fragment that is a Fab, single V_(H) domain, asingle chain variable fragment (scFv), a multivalent scFv, a bispecificscFv or an scFv-CH3 dimer.

16. The method of any of embodiments 1-15, wherein the irradiation iscarried out between or between about 30 minutes and 96 hours afteradministering the conjugate.

17. The method of any of embodiments 1-16, wherein the lesion isirradiated at a wavelength of 690±50 nm or at a wavelength of or about690±20 nm.

18. The method of any of embodiments 1-17, wherein the lesion isirradiated at a dose of from or from about 2 J cm⁻² to about 400 J cm⁻²or from or from about 2 J/cm fiber length to about 500 J/cm fiberlength.

19. The method of any of embodiments 1-18, wherein:

the lesion is irradiated at a dose of at least or at least about 2 Jcm⁻², 5 J cm⁻², 10 J cm⁻², 25 J cm⁻², 50 J cm⁻², 75 J cm⁻², 100 J cm⁻²,150 J cm⁻², 200 J cm⁻², 300 J cm⁻², 400 J cm⁻², or 500 J cm⁻²; or

the lesion is irradiated at a dose of at least or at least about 2 J/cmfiber length, 5 J/cm fiber length, 10 J/cm fiber length, 25 J/cm fiberlength, 50 J/cm fiber length, 75 J/cm fiber length, 100 J/cm fiberlength, 150 J/cm fiber length, 200 J/cm fiber length, 250 J/cm fiberlength, 300 J/cm fiber length, 400 J/cm fiber length or 500 J/cm fiberlength.

20. The method of any of embodiments 1-19, wherein the phthalocyaninedye has a maximum absorption wavelength from or from about 600 nm toabout 850 nm.

21. The method of any of embodiments 1-20, wherein the phthalocyaninedye is linked directly or indirectly to the targeting molecule.

22. The method of any of embodiments 1-21, wherein the phthalocyaninedye comprises the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

23. The method of any of embodiments 1-22, wherein the phthalocyaninedye comprises the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

24. The method of any of embodiments 1-23, wherein the phthalocyaninedye comprises IRDye 700DX (IR700).

25. The method of any of embodiments 1-24, wherein the cell surfaceprotein is selected from among ACTHR, endothelial cell Anxa-1,aminopetidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin,alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN,APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B-cell receptor BB1, BB2, BB4,calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10,CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52,CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA(CarcinoEmbryonic Antigen), CGRP, chemokine receptors, cell-surfaceannexin-1, cell-surface plectin-1, Cripto-1, CRLR, CXCR2, CXCR4, DCC,DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, Endosialin, EP2, EP4,EpCAM, EphA2, ET receptors, Fibronectin, Fibronectin ED-B, FGFR,frizzled receptors, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1receptor, G-protein coupled receptors of the Family A (Rhodopsin-like),G-protein coupled receptors of the Family B (Secretin receptor-like)like), G-protein coupled receptors of the Family C (MetabotropicGlutamate Receptor-like), GD2, GP100, GP120, Glypican-3, hemagglutinin,Heparin sulfates, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7antigens, hTERT, IL11-R, IL-13R, ITGAM, Kalikrien-9, Lewis Y, LHreceptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE 4, MART 1,MC1R, Mesothelin, MUC1, MUC16, Neu (cell-surface Nucleolin), Neprilysin,Neuropilin-1, Neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1,NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32(p32/gC1q-R/HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFGreceptors, PDT, Protease-cleaved collagen IV, proteinase 3, prohibitin,protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g.,P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patched, RET receptor, plexins,smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEMs,T-cell CD3 Receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B,Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1,TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptors(VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, and VEGF-3 or FLT-4),voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, andY5.

26. The method of any of embodiments 1-25, wherein the cell surfaceprotein is selected from among HER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2Rareceptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5,CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y,TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2receptor, vascular endothelial growth factor (VEGF), CD30, EpCAM, EphA2,Glypican-3, gpA33, mucins, CAIX, PSMA, folate-binding protein,gangliosides (such as GD2, GD3, GM1 and GM2), VEGF receptor (VEGFR),integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1,TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44,CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen, IgE, MUC-1, nuC242, PEMantigen, metalloproteinases, Ephrin receptor, Ephrin ligands, HGFreceptor, CXCR4, CXCR4, Bombesin receptor, and SK-1 antigen.

27. The method of any of embodiments 1-26, wherein the cell surfaceprotein is selected from among CD25, PD-1 (CD279), PD-L1 (CD274, B7-H1),PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3 (HAVCR2), 4-1BB (CD137,TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1, Galectin 9, BTLA, CD160,VISTA (PD1 homologue), B7-H4 (VCTN1), CD80 (B7-1), CD86 (B7-2), CD28,HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT, CD96, Galectin 3, CD40,CD40L, CD70, LIGHT (TNFSF14), HVEM (TNFRSF14), B7-H3 (CD276), Ox40L(TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS (CD278), ICOSL, KIR, GALS,NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, Butyrophilin family,CD48, CD244, Siglec family, CD30, CSF1R, MICA (MEW class Ipolypeptide-related sequence A), MICB (MEW class I polypeptide-relatedsequence B), NKG2D, KIR family (Killer-cell immunoglobulin-likereceptor, LILR family (Leukocyte immunoglobulin-like receptors, CD85,ILTs, LIRs), SIRPA (Signal regulatory protein alpha), CD47 (IAP),Neuropilin 1 (NRP-1), a VEGFR, and VEGF.

28. The method of any of embodiments 1-27, wherein the antibody or anantigen-binding antibody fragment is selected from among cetuximab,panitumumab, zalutumumab, nimotuzumab, Tositumomab (Bexxar®), Rituximab(Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin), Daclizumab(Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fab fragment,OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab (Avastin®),Basiliximab, nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559,MPDL3280A, ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525,urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab,SEA-CD40, CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916,AMP-224, PDR001, MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab(CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201,AGX-115, Emactuzumab, CC-90002 and MNRP1685A or is an antigen-bindingantibody fragment thereof.

29. The method of any of embodiments 1-28, wherein the conjugate isselected from among cetuximab-IR700, panitumumab-IR700,zalutumumab-IR700, nimotuzumab-IR700, Tositumomab-IR700,Rituximab-IR700, Ibritumomab tiuxetan-IR700, Daclizumab-IR700,Gemtuzumab-IR700, Alemtuzumab-IR700, CEA-scan Fab fragment-IR700,OC125-IR700, ab75705-IR700, B72.3-IR700, Bevacizumab-IR700,Basiliximab-IR700, nivolumab-IR700, pembrolizumab-IR700,pidilizumab-IR700, MK-3475-IR700, BMS-936559-IR700, MPDL3280A-IR700,ipilimumab-IR700, tremelimumab-IR700, IMP321-IR700, BMS-986016-IR700,LAG525-IR700, urelumab-IR700, PF-05082566-IR700, TRX518-IR700,MK-4166-IR700, dacetuzumab-IR700, lucatumumab-IR700, SEA-CD40-IR700,CP-870-IR700, CP-893-IR700, MED16469-IR700, MED16383-IR700,MED14736-IR700, MOXR0916-IR700, AMP-224-IR700, PDR001-IR700,MSB0010718C-IR700, rHIgM12B7-IR700, Ulocuplumab-IR700, BKT140-IR700,Varlilumab-IR700, ARGX-110-IR700, MGA271-IR700, lirilumab-IR700,IPH2201-IR700, AGX-115-IR700, Emactuzumab-IR700, CC-90002-IR700 andMNRP1685A-IR700.

30. The method of embodiment 29, wherein the targeting molecule is anantibody that is cetuximab or is an antigen-binding antibody fragmentthereof or the conjugate is cetuximab-IR700.

31. The method of embodiment 30, wherein the average area under theplasma conjugate concentration-time curve from time 0 to infinity(AUC[0-inf]) for a patient population after administration of theconjugate is between or between about 500 μg/mL*h and 18,000 μg/mL*h,between or between about 500 μg/mL*h and 10,000 μg/mL*h, between orbetween about 500 μg/mL*h and 5,000 μg/mL*h, or between or between about500 μg/mL*h and 2,500 μg/mL*h.

32. The method of embodiment 30, wherein the average area under theplasma conjugate concentration-time curve from time 0 to 24 hours(AUC[0-24]) for a patient population after administration of theconjugate is between or between about 500 μg/mL*h and 8,000 μg/mL*h,between or between about 500 μg/mL*h and 5,000 μg/mL*h, between orbetween about 500 μg/mL*h and 2,000 μg/mL*h or between or between about1000 μg/mL*h and 4,000 μg/mL*h.

33. The method of any of embodiments 30-32, wherein:

the conjugate is administered in a dosage range that between or betweenabout 75 mg/m² (body surface area of the subject) and 1500 mg/m²,between or between about 75 mg/m² and 1000 mg/m², between or betweenabout 75 mg/m² and 500 mg/m² or between or between about 75 mg/m² and225 mg/m²; or

is at least about or is about 160 mg/m², 320 mg/m², 640 mg/m² or 1280mg/m².

34. A method of treating a disease lesion in a subject, comprising:

a) intravenously administering to a subject having a lesion associatedwith a disease or condition a cetuximab-IR700 conjugate, wherein theconjugate is administered in an amount that is or is about 640 mg/m²;and

b) after administering the conjugate, irradiating the lesion at awavelength of 690±20 nm at a dose of at least or about at least or about50 J cm⁻² or 100 J/cm of fiber length, thereby treating the disease orcondition in the subject.

35. The method of embodiment 34, wherein the conjugate is administeredin a dosing schedule in which:

the administration of the conjugate is performed only one time as asingle injection or infusion; or

the dosing schedule does not comprise a subsequent dose of theconjugate; or

the dosing schedule does not comprise a subsequent dose of themacromolecule that is not so conjugated.

36. The method of any of embodiments 1-35, wherein the irradiation iscarried out 24 hours±3 hours after administering the conjugate.

37. The method of any of embodiments 34-36, wherein the lesion is atumor and the disease or condition is a tumor or a cancer.

38. The method of any of embodiments 1-37, wherein the lesion is a tumorthat is a superficial tumor.

39. The method of embodiment 38, wherein the tumor is less than 10 mmthick.

40. The method of embodiment 38 or embodiment 39, wherein irradiation iscarried out using a microlens-tipped fiber for surface illumination.

41. The method of any of embodiments 1-40, wherein the light irradiationdose is from or from about 5 J/cm² to about 200 J/cm².

42. A method for treating a superficial tumor with photoimmunotherapy,comprising illuminating an superficial tumor in a subject with amicrolens-tipped fiber for surface illumination with a light dose offrom or from about 5 J/cm² to about 200 J/cm², wherein the tumor isassociated with a phototoxic agent comprising a targeting molecule boundto a cell surface molecule of the tumor.

43. The method embodiment 41 or embodiment 42, wherein the lightirradiation dose is or is about 50 J/cm².

44. The method of any of embodiments 1-40, wherein the lesion is a tumorthat is an interstitial tumor.

45. The method of embodiment 44, wherein the tumor is greater than 10 mmdeep or is a subcutaneous tumor.

46. The method of embodiment 44 or embodiment 45, wherein irradiation iscarried out using cylindrical diffusing fibers comprising a diffuserlength of 0.5 cm to 10 cm and spaced 1.8±0.2 cm apart.

47. The method of any of embodiments 1-37 and 44-46, wherein the lightirradiation dose is from or from about 20 J/cm fiber length to about 500J/cm fiber length.

48. A method for treating an interstitial tumor with photoimmunotherapy,comprising illuminating an interstitial tumor in a subject withcylindrical diffusing fibers comprising a diffuser length of 0.5 cm to10 cm and spaced 1.8±0.2 cm apart with a light dose of or about 100 J/cmfiber length or with a fluence rate of or about 400 mW/cm, wherein thetumor is associated with a phototoxic agent comprising a targetingmolecule bound to a cell surface molecule of the tumor.

49. The method of embodiment 47 or embodiment 48, wherein the lightirradiation dose is from or from about 50 J/cm fiber length to about 300J/cm fiber length.

50. The method of any of embodiments 47-49, wherein the lightirradiation dose is or is about 100 J/cm fiber length.

51. The method of any of embodiments 48-50, wherein the tumor is greaterthan 10 mm deep or is a subcutaneous tumor.

52. The method of any of embodiments 47-51, wherein the cylindricaldiffusing fibers are placed in a catheter positioned in the tumor1.8±0.2 cm apart.

53. The method of embodiment 52, wherein the catheter is opticallytransparent.

54. The method of any of embodiments 42, 43 and 48-53, wherein greaterthan 6 hours prior to illuminating the tumor, the subject has beenadministered the phototoxic agent comprising the targeting molecule,wherein the phototoxic agent associates with the tumor.

55. The method of embodiment 54, wherein the phototoxic agent has beenpreviously administered to the subject greater than or greater thanabout 12 hours, 24 hours, 26 hours, 48 hours, 72 hours or 96 hours priorto illuminating the tumor.

56. The method of any of embodiments 42, 43 and 48-55, wherein thephototoxic agent is a phthalocyanine dye-targeting molecule conjugate.

57. The method of embodiment 56, wherein the phthalocyanine dye isIR700.

58. The method of any of embodiments 1-41, 44-47, 49, 50 and 52-54,wherein the dosing schedule is repeated, whereby steps (a) and (b) arerepeated.

59. The method of embodiment 58, wherein the dosing schedule is repeatedif residual lesion remains after a prior treatment with the conjugate.

60. The method of embodiment 58 or embodiment 59, comprising assessingthe subject for the presence of a residual lesion and if residual lesionremains repeating the dosing schedule.

61, The method of any of embodiments 58-60, wherein the dosing scheduleis repeated if a residual lesion remains at a time that is more than orabout or 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 yearafter initiation of the prior administration of the conjugate.

62. The method of any of embodiments 58-61, wherein the dosing scheduleis repeated if a residual lesion remains at or about 4 weeks afterinitiation of the prior administration of the conjugate.

63. The method of any of embodiments 1-62, wherein the conjugatecomprises 1 to 100, 1 to 10 or 2 to 5 phthalocyanine dye molecules permacromolecule.

64. The method of any of embodiments 1-63, wherein the method does notcomprise administration of an additional therapeutic agent oranti-cancer treatment.

65. The method of any of embodiments 1-63, wherein the method comprisesadministration of an additional therapeutic agent or anti-cancertreatment.

66. The method of embodiment 65, wherein the anti-cancer treatmentcomprises radiation therapy.

67. The method of embodiment 66, wherein the additional therapeuticagent is an anti-cancer agent or an immune modulating agent, with immunemodulating agent optionally is a cell-based (e.g. combination treatmentwith immune cells such as dendritic cells) or non-cell based immunemodulating agents.

68. The method of embodiment 67, wherein the additional therapeuticagent is an immune modulating agent that is an immune checkpointinhibitor.

69. The method of embodiment 68, wherein the immune checkpoint inhibitorspecifically binds a molecule selected from among CD25, PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

70. The method of embodiment 68 or embodiment 69, wherein the immunecheckpoint inhibitor is and antibody or antigen-binding fragment, asmall molecule or a polypeptide.

71. The method of any of embodiments 68-70, wherein the immunecheckpoint inhibitor is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof.

72. The method of any of embodiments 67-71, wherein the additionaltherapeutic agent is administered prior to irradiating the lesion ortumor.

73. The method of embodiment 72, wherein the additional therapeuticagent is administered greater than or greater than about 30 minutes, 1hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 96 hours, oneweek, two weeks, three weeks or one month prior to irradiating thetumor.

74. The method of any of embodiments 67-73, comprising continuedadministration of the additional therapeutic agent subsequent to theirradiation three times a week, two times a week, once every week, onceevery two weeks, once every three weeks or once a month.

75. A method of treating a tumor in a subject comprising:

a) administering to a subject an immune modulating agent;

b) administering to the subject a therapeutically effective amount of aconjugate comprising a phthalocyanine dye linked to a targeting moleculecapable of binding to a molecule on the surface of a cell present in themicroenvironment of a tumor; and

c) greater than 12 hours after administering the immune modulatingagent, irradiating the tumor at a wavelength that renders the conjugatecytotoxic, thereby treating the tumor.

76. The method of embodiment 75, wherein the immune modulating agent isadministered greater than or greater than about 24 hours, 48 hours, 96hours, one week, two weeks, three weeks or one month prior toirradiating the tumor.

77. The method of embodiment 75 or embodiment 76, wherein the conjugatebinds to a protein on the surface of a cell present in themicroenvironment of the tumor.

78. The method of any of embodiments 75-77, wherein step c) ofirradiating the tumor is carried out either i) after administration ofthe immune modulating agent and after administration of the conjugate orii) only after administration of the conjugate.

79. The method of any of embodiments 67-78, wherein the conjugate isadministered prior to, simultaneously or subsequently to administrationof the immune-modulating agent.

80. The method of any of embodiments 67-79, wherein the conjugate isadministered after administering the immune modulating agent but priorto irradiating the tumor.

81. The method of any of embodiments 67-80, wherein the conjugate isadministered from or from about 12 hours to 48 hours prior toirradiating the tumor and the immune modulating agent is administeredfrom or from about 12 hours to about 1 month prior to irradiating thetumor.

82. The method of any of embodiments 75-81, wherein the immunemodulating agent is an immune checkpoint inhibitor.

83. The method of embodiment 82, wherein the immune checkpoint inhibitorspecifically binds a molecule selected from among CD25, PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

84. The method of embodiment 82 or embodiment 83, wherein the immunecheckpoint inhibitor is and antibody or antigen-binding fragment, asmall molecule or a polypeptide.

85. The method of any of embodiments 82-84, wherein the immunecheckpoint inhibitor is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing.

86. The method of any of embodiments 75-81, wherein the immunemodulating agent that is a demethylating agent that upregulatesexpression of a tumor associated antigen (TAA) or is a cytokine.

87. The method of any of embodiments 75-86, comprising continuedadministration of the immune modulating agent subsequent to theirradiation three times a week, two times a week, once every week, onceevery two weeks, once every three weeks or once a month.

88. A method of treating a tumor in a subject comprising:

a) administering to a subject an immune modulating agent that enhancesthe expression of a molecule on the surface of a cell present in themicroenvironment of the tumor;

b) administering to the subject a therapeutically effective amount of aconjugate comprising a phthalocyanine dye linked to a targeting moleculethat is capable of binding to the cell surface molecule; and

c) greater than 5 minutes after administering the conjugate, irradiatingthe tumor at a wavelength that renders the conjugate cytotoxic, therebytreating the tumor.

89. The method of embodiment 88, wherein the immune modulating agent isa cytokine or is an agent that induces increased expression of acytokine in the tumor microenvironment.

90. The method of embodiment 88 or embodiment 89, wherein the cytokineis interferon gamma.

91. The method of any of embodiments 88-90, wherein the molecule on thesurface of the cells is selected from CD25, PD-1, PD-L1, PD-L2, CTLA-4,LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3,B7-H4, BTLA, HVEM, CD28 and VISTA.

92. The method of any of embodiments 88-91, wherein the molecule on thesurface of the cell is PD-L1.

93. The method of any of embodiments 88-92, wherein the targetingmolecule is an immune checkpoint inhibitor.

94. The method of any of embodiments 88-93, wherein the targetingmolecule is an antibody or antibody fragment, a small molecule or apolypeptide.

95. The method of any of embodiments 88-94, wherein the targetingmolecule is selected from among nivolumab, pembrolizumab, pidilizumab,MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31,BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing.

96. A method of treating a tumor in a subject comprising:

a) administering to a subject a conjugate comprising a phthalocyaninedye linked to a targeting molecule capable of binding a cell surfacemolecule on a cell in the microenvironment of the tumor;

b) greater than 5 minutes after administering the conjugate, irradiatingthe tumor at a wavelength that renders the conjugate cytotoxic, whereinthe treatment of the tumor with the conjugate followed by lightirradiation increases the presence of immunosuppressive cells in thetumor or increases the expression of immunosuppressive markers at thetumor; and

c) administering to the subject a therapeutically effective amount of animmune modulating agent capable of reducing the amount or activity ofimmunosuppressive cells in the tumor or capable of blocking the activityof the immunosuppressive marker.

97. The method of embodiment 96, wherein the phthalocyanine dye is afirst dye and the immune modulating agent comprises a conjugatecomprising a second phthalocyanine dye conjugated to an immunemodulating agent capable of binding to the immunosuppressive cell.

98. The method of embodiment 97, wherein the first and secondphthalocyanine dye is the same or different.

99. The method of any of embodiments 96-98, wherein the immunemodulating agent is an immune checkpoint inhibitor.

100. The method of any of embodiments 96-99, wherein the immunemodulating agent specifically binds a molecule selected from among CD25,PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L,OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

101. The method of any of embodiments 96-100, wherein the immunemodulating agent is an antibody or antibody fragment, a small moleculeor a polypeptide.

102. The method of any of embodiments 96-101, wherein the immunemodulating agent is not an anti-CTLA4 antibody.

103. The method of any of embodiments 96-102, wherein the immunemodulating agent is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing.

104. A method of treating a tumor in a subject comprising:

a) administering to a subject a conjugate comprising a phthalocyaninedye linked to a targeting macromolecule capable of binding to a moleculeon the surface of a cell present in the microenvironment of the tumor;

b) greater than 5 minutes after administering the conjugate, irradiatingthe tumor at a wavelength that renders the conjugate cytotoxic, whereinthe treatment of the tumor with the conjugate followed by lightirradiation primes activation of immune cells; and

c) administering to the subject a therapeutically effective amount of animmune modulating agent capable of increasing the activity of the immunecell.

105. The method of embodiment 104, wherein the immune cell is an antigenpresenting cell.

106. The method of embodiment 105, wherein the immune cell is adendritic cell.

107. The method of any of embodiments 104-106, wherein the immunemodulating agent is selected from among GM-CSF, CpG-ODN (CpGoligodeoxynucleotides), lipopolysaccharide (LPS), monophosphoryl lipid A(MPL), alum, recombinant Leishmania polyprotein, imiquimod, MF59, polyI:C, poly A:U, type 1 IFN, Pam3Cys, Pam2Cys, complete freund's adjuvant(CFA), alpha-galactosylceramide, RC-529, MDF2(3, Loxoribine, anti-CD40agonist, SIRPa antagonist, AS04, AS03, Flagellin, Resiquimod, DAP(diaminopimelic acid), MDP (muramyl dipeptide) CAF01 (cationic adjuvantformulation-01), antrhacyclins (doxorubicin, mitoxantron), BK channelagonists, bortezomib, botrtezomib plus mitocycin C plus hTert-Ad,Cardiac glycosides plus non-Immunogenic cell death inducers,cyclophosphamide, GADD34/PP1 inhibitors plus mitomycin, LV-tSMAC, andoxaliplatin.

108. The method of any of embodiments 104-107, wherein the immunemodulating agent is a Toll-like receptor (TLR) agonist, an adjuvant or acytokine.

109. The method of embodiment 108, wherein the immune modulating agentis a TLR agonist and the TLR agonist is TLR agonist is a TLR4 agonist, aTLR7 agonist, a TLR8 agonist, or a TLR9 agonist.

110. The method of embodiment 108 or embodiment 109, wherein the TLRagonist is selected from among triacylated lipoprotein, diacylatedlipopeptide, lipoteichoic acid, peptidoglycan, zymosan, Pam3CSK4, dsRNA,polyI:C, Poly G10, Poly G3, CpG, 3M003, flagellin, lipopolysaccharide(LPS) Leishmania homolog of eukaryotic ribosomal elongation andinitiation factor 4a (LeIF), MEDI9197, SD-101, and imidazoquinoline TLRagonists.

111. The method of any of embodiments 104-107, wherein the immunemodulating agent is a cytokine and the cytokine is IL-4, TNF-α, GM-CSFor IL-2.

112. The method of any of embodiments 96-111, wherein the immunemodulating agent is administered prior to, simultaneously with or afterthe irradiation.

113. The method of embodiment 112, wherein the immune modulating agentis administered no more than 5 minutes, 30 minutes, 60 minutes, 2 hours,6 hours, 12 hours or 24 hours after the irradiation.

114. The method of any of embodiments 75-113, wherein the targetingmolecule binds to molecule or protein directly or indirectly.

115. The method of embodiment 114, wherein the targeting molecule is asecond binding molecule that binds to a first binding molecule, saidfirst binding molecule being capable of binding to the molecule orprotein.

116. The method of embodiment 114 or embodiment 115, wherein thetargeting molecule is a secondary antibody.

117. The method of any of embodiments 75-116, wherein the phthalocyaninedye has a maximum absorption wavelength from or from about 600 nm toabout 850 nm.

118. The method of any of embodiments 75-117, wherein the phthalocyaninedye is covalently or non-covalently linked to the targeting molecule.

119. The method of any of embodiments 75-118, wherein the phthalocyaninedye comprises a linker comprising a reactive group for attachment of thedye to the targeting molecule.

120. The method of embodiment 119, wherein the phthalocyanine dyecomprises the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

121. The method of embodiment 119 or embodiment 120, wherein thephthalocyanine dye comprises the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

122. The method of any of embodiments 75-121, wherein the phthalocyaninedye comprises IRDye 700DX (IR700).

123. The method of any of embodiments 75-122, wherein the conjugate isadministered at a dose from or from about 50 mg/m² to about 5000 mg/m²,from about 250 mg/m² to about 2500 mg/m², from about 750 mg/m² to about1250 mg/m² or from about 100 mg/m² to about 1000 mg/m².

124. The method of any of embodiments 8-33 and 37-123, wherein the tumoris a cancer.

125. The method of embodiment 124, wherein the cancer is a cancerlocated at the head and neck, breast, liver, colon, ovary, prostate,pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus,peritoneum, or lung.

126. The method of any of embodiments 8-33 and 37-125, wherein the tumoris a sarcoma or carcinoma.

127. The method of embodiment 126, wherein the tumor is a carcinoma thatis a squamous cell carcinoma, basal cell carcinoma or adenocarcinoma.

128. The method of embodiment 127, wherein the tumor is a carcinoma thatis a carcinoma of the bladder, pancreas, colon, ovary, lung, breast,stomach, prostate, cervix, esophagus or head and neck.

129. The method of any of embodiments 75-128, wherein the tumor isirradiated at a wavelength of 600 nm to 850 nm at a dose of at least 1 Jcm⁻² or at least 1 J/cm fiber length.

130. The method of any of embodiments 75-129, wherein the tumor isirradiated at a wavelength of 690 nm±50 nm or at a wavelength of orabout 690±20 nm.

131. The method of any of embodiments 1-130, wherein the method reducesthe size or volume of the tumor by at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80% at least 90% or morewithin one month of the irradiation compared to the size or volume ofthe tumor prior to the administration and irradiation.

132. The method of any of embodiments 1-131, which, in a population oftreated subjects, effects an improvement of a disorder- orcancer-related parameter compared to a similarly situated population ofsubjects treated with the antibody or antigen-binding antibody fragmentthat is not conjugated, wherein the parameter is selected from one ormore of: a) objective response rate (ORR); b) progression free survival(PFS); c) overall survival (OS); d) reduction in toxicity; e) tumorresponse; of f) quality of life.

133. The method of embodiment 132, wherein the parameter is improved byat least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100% ormore.

134. The method of any of embodiments 1-133, which, in a population oftreated subjects, effects an objective response rate (ORR) of at least15%, at least 25%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% or more.

135. The method of any of embodiments 1-133, wherein the phthalocyaninedye is a first dye and the conjugate further comprises a secondfluorescent dye linked to the macromolecule that is different than thefirst dye.

136. The method of embodiment 135, wherein irradiating the lesion ortumor emits a fluorescence signal from the second fluorescent dye toeffect detection of the presence of the conjugate at the lesion or tumorin the subject.

137. The method of embodiment 135 or embodiment 136, further comprisingimaging the lesion or tumor in the subject by irradiating orilluminating the tumor at a wavelength capable of being absorbed by thesecond dye.

138. The method of any of embodiments 135-137, wherein the secondfluorescent dye exhibits one or more spectral properties selected fromamong fluorescent quantum yield in water, extinction coefficient, Stokesshift, absorption and emission at long wavelength and photostabilitythat is greater compared to the corresponding spectral property of thefirst dye.

139. The method of any of embodiments 135-138, wherein the first dye isIR700.

140. The method of any of embodiments 135-139, wherein the second dye isnot IR700.

141. The method of any of embodiments 135-140, wherein the second dye isselected from among hydroxycoumarin, Cascade Blue, Dylight 405, PacificOrange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor 488,BODIPY 493, 2.7-Diochlorofluorescien, ATTO 488, Chromeo 488, Dylight488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF 555,Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555, Dylight550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine, Texas Red,Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor 647, APC,ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, Alexa Fluor 680,IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750, Dylight 755,IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800, Qdot® 525,Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

142. The method of any of embodiments 135-141, wherein the first dye isIR700 and the conjugate comprises 1 to 10 or 1 to 5 second dye moleculesper macromolecule.

143. The method of any of embodiments 135-142, wherein the second dyeexhibits a Stokes shift that is greater than 15 nm, greater than 20 nm,greater than 30 nm, greater than 40 nm, greater than 50 nm, greater than60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm orgreater than 100 nm.

144. The method of any of embodiments 135-143, wherein the second dyehas a quantum yield in water that is greater than 10%, greater than 15%,greater than 20% or greater than 25%, greater than 30%, greater than40%, greater than 50% or greater.

145. The method of any of embodiments 135-144, wherein the second dyehas an absorption and emission wavelength in the spectrum between orbetween about 650 nm and 950 nm, between or between about 700 nm and1000 nm, or between or between about 1000 nm and 1700 nm.

146. The method of any of embodiments 135-145, wherein the first dye andsecond dye do not exhibit an overlapping emission and absorptionspectra.

147. The method of any of embodiments 135-1146, wherein the second dyeis selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755,IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800.

148. The method of any of embodiments 135-147, wherein the second dye isAlexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

149. The method of any of embodiments 1-148, wherein the irradiating orilluminating is performed with a device selected from among a hand-heldultraviolet lamp, a mercury lamp, a xenon lamp, a laser, a laser diodeor an LED imaging device.

150. The method of embodiment 149, wherein the imaging device comprisesa near-infrared (NIR) diode.

151. A composition, comprising a conjugate comprising a phthalocyaninedye linked to an antibody or antigen-binding antibody fragment thatbinds to a molecule on the surface of a cell present in themicroenvironment of a lesion, wherein the composition is formulated forsingle dosage administration of the conjugate in an amount that isbetween or between about 100 mg and 2000 mg.

152. The composition of embodiment 151, wherein the composition isformulated for single dosage administration of an amount between orbetween about 100 mg and 500 mg, between or between about 200 mg and 400mg. 153. The composition of embodiment 151 or embodiment 152, whereinthe composition is formulated for single dosage administration of anamount between or between about 500 mg and 1500 mg, 800 mg and 1200 mg,or 1000 mg and 1500 mg.

154. The composition of any of embodiments 151-153, wherein:

the volume of the composition is between or between about 10 mL and 1000mL, or 50 mL and 500 mL; or the volume of the composition is at least 10mL, 20 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 150 mL, 200 mL, 250 mL,300 mL, 400 mL, 500 mL or 1000 mL.

155. An article of manufacture, comprising:

a plurality of sealable containers, each individually comprising afraction of a single administration dose of a composition comprising aconjugate comprising a phthalocyanine dye linked to an antibody orantigen-binding antibody fragment that binds to a molecule on thesurface of a cell present in the microenvironment of a lesion, whereinthe combined amount of the conjugate in the plurality of sealablecontainers is between or between about 100 mg and 1500 mg;

packaging material; and

a label or package insert comprising instructions for combining thecontents of the plurality of vials to prepare a single dosageformulation of the composition.

156. The article of manufacture of embodiment 155, wherein the combinedamount of the conjugate in the plurality of sealable containers isbetween or between about 100 mg and 1200 mg.

157. The article of manufacture of embodiment 155 or embodiment 156,wherein the combined amount of the conjugate in the plurality ofsealable container is between or between about 100 mg and 500 mg,between or between about 200 mg and 400 mg, between or between about 500mg and 1500 mg, between or between about 800 mg and 1200 mg or betweenor between about 1000 mg and 1500 mg.

158. The composition of any of embodiments 151-154 or the article ofmanufacture of any of embodiments 155-157, wherein the lesion is atumor.

159. A conjugate, comprising a phthalocyanine dye linked to an antibodyor antigen-binding fragment that is an immune modulating agent.

160. The conjugate of embodiment 159, wherein the immune modulatingagent is an immune checkpoint inhibitor.

161. The conjugate of embodiment 159 or embodiment 160, wherein theimmune modulating agent is an antibody or antigen binding fragment thatbinds to the surface of a tumor, tumor cell or cancer cell.

162. The conjugate of any of embodiments 159-161, wherein the immunemodulating agent specifically binds a molecule selected from among CD25,PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L,OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

163. The conjugate of any of embodiments 159-162, wherein the immunemodulating agent is selected from among nivolumab, pembrolizumab,pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab,IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MED14736, MOXR0916, AMP-224, and MSB001078C,or is an antigen-binding fragment thereof of any of the foregoing.

164. The conjugate of any of embodiments 159-163, wherein the immunemodulating agent is an antibody or antibody fragment that binds toPD-L1.

165. The conjugate of embodiment 164, wherein the immune modulatingagent is an antibody selected from BMS-935559, MEDI4736, MPDL3280A andMSB0010718C, or an antigen-binding fragment thereof.

166. A conjugate, comprising a targeting molecule linked to at least afirst and second fluorescent dye, wherein the first fluorescent dye is aphthalocyanine dye capable of exhibiting phototoxicity.

167. The conjugate of embodiment 166, comprising the formula:

[D₁-(L₁)_(n)]_(p)-A-[(L₂)_(n)-D₂]_(o), wherein:

A is a targeting molecule that can bind to a molecule on the surface ofa cell;

L₁ and L₂ are each an independently selected linker, which can be thesame or different;

n and m are independently 1 or 2;

D₁ is a first dye that is the phthalocyanine dye capable of exhibitingphototoxicity;

D₂ is a second dye that is a fluorescent dye, wherein D₂ is differentthan D₁;

p is 1 to 10; and

o is 1 to 10.

168. The conjugate of embodiment 166 or embodiment 167, wherein thetargeting molecule is an antibody or an antigen-binding antibodyfragment.

169. The conjugate of any of embodiments 166-168, wherein the cellsurface molecule comprises an antigen, a polypeptide, a lipid, or acarbohydrate or a combination of these molecules.

170. The conjugate of any of embodiments 166-169, wherein the cellsurface molecule is selected from among ACTHR, endothelial cell Anxa-1,aminopetidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin,alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN,APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B-cell receptor BB1, BB2, BB4,calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10,CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52,CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA(CarcinoEmbryonic Antigen), CGRP, chemokine receptors, cell-surfaceannexin-1, cell-surface plectin-1, Cripto-1, CRLR, CXCR2, CXCR4, DCC,DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR1, Endosialin, EP2, EP4,EpCAM, EphA2, ET receptors, Fibronectin, Fibronectin ED-B, FGFR,frizzled receptors, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1receptor, G-protein coupled receptors of the Family A (Rhodopsin-like),G-protein coupled receptors of the Family B (Secretin receptor-like)like), G-protein coupled receptors of the Family C (MetabotropicGlutamate Receptor-like), GD2, GP100, GP120, Glypican-3, hemagglutinin,Heparin sulfates, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7antigens, hTERT, IL11-R, IL-13R, ITGAM, Kalikrien-9, Lewis Y, LHreceptor, LHRH-R, LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE 4, MART 1,MC1R, Mesothelin, MUC1, MUC16, Neu (cell-surface Nucleolin), Neprilysin,Neuropilin-1, Neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1,NY-ESO-1, OT-R, mutant p53, p97 melanoma antigen, NTR2, NTR3, p32(p32/gC1q-R/HABP1), p75, PAC1, PAR1, Patched (PTCH), PDGFR, PDFGreceptors, PDT, Protease-cleaved collagen IV, proteinase 3, prohibitin,protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g.,P2X1-5), mutant Ras, RAMP1, RAMP2, RAMP3 patched, RET receptor, plexins,smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEMs,T-cell CD3 Receptor, TAG72, TGFBR1, TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B,Trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1,TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3), TSH receptor, VEGF receptors(VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, and VEGF-3 or FLT-4),voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor 1, Y1, Y2, Y4, andY5.

171. The conjugate of any of embodiments 166-170, wherein the cellsurface molecule is selected from among HER1/EGFR, HER2/ERBB2, CD20,CD25 (IL-2Ra receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3,CEACAM5, CEACAM6, cancer antigen 125 (CA125), alpha-fetoprotein (AFP),Lewis Y, TAG72, Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1,CTLA-4, IL-2 receptor, vascular endothelial growth factor (VEGF), CD30,EpCAM, EphA2, Glypican-3, gpA33, mucins, CAIX, PSMA, folate-bindingprotein, gangliosides (such as GD2, GD3, GM1 and GM2), VEGF receptor(VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3,TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18,CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen, IgE, MUC-1, nuC242,PEM antigen, metalloproteinases, Ephrin receptor, Ephrin ligands, HGFreceptor, CXCR4, CXCR4, Bombesin receptor, and SK-1 antigen.

172. The conjugate of any of embodiments 166-171, wherein the cellsurface molecule is selected from among CD25, PD-1 (CD279), PD-L1(CD274, B7-H1), PD-L2 (CD273, B7-DC), CTLA-4, LAG3 (CD223), TIM3(HAVCR2), 4-1BB (CD137, TNFRSF9), CXCR2, CXCR4 (CD184), CD27, CEACAM1,Galectin 9, BTLA, CD160, VISTA (PD1 homologue), B7-H4 (VCTN1), CD80(B7-1), CD86 (B7-2), CD28, HHLA2 (B7-H7), CD28H, CD155, CD226, TIGIT,CD96, Galectin 3, CD40, CD40L, CD70, LIGHT (TNFSF14), HVEM (TNFRSF14),B7-H3 (CD276), Ox40L (TNFSF4), CD137L (TNFSF9, GITRL), B7RP1, ICOS(CD278), ICOSL, KIR, GALS, NKG2A (CD94), GARP, TL1A, TNFRSF25, TMIGD2,BTNL2, Butyrophilin family, CD48, CD244, Siglec family, CD30, CSF1R,MICA (MHC class I polypeptide-related sequence A), MICB (MHC class Ipolypeptide-related sequence B), NKG2D, KIR family (Killer-cellimmunoglobulin-like receptor, LILR family (Leukocyte immunoglobulin-likereceptors, CD85, ILTs, LIRs), SIRPA (Signal regulatory protein alpha),CD47 (IAP), Neuropilin 1 (NRP-1), a VEGFR, and VEGF.

173. The conjugate of any of embodiments 166-172, wherein themacromolecule is an antibody or an antigen-binding antibody fragmentthat is selected from among cetuximab, panitumumab, zalutumumab,nimotuzumab, Tositumomab (Bexxar®), Rituximab (Rituxan, Mabthera),Ibritumomab tiuxetan (Zevalin), Daclizumab (Zenapax), Gemtuzumab(Mylotarg), Alemtuzumab, CEA-scan Fab fragment, OC125 monoclonalantibody, ab75705, B72.3, Bevacizumab (Avastin®), Basiliximab,nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A,ipilimumab, tremelimumab, IMP321, BMS-986016, LAG525, urelumab,PF-05082566, TRX518, MK-4166, dacetuzumab, lucatumumab, SEA-CD40,CP-870, CP-893, MED16469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001,MSB0010718C, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127),ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115,Emactuzumab, CC-90002 and MNRP1685A or is an antigen-binding antibodyfragment thereof.

174. The conjugate of any of embodiments 166-173, wherein the targetingmolecule is not or does not comprise a nanocarrier.

175. The conjugate of any of embodiments 166-174, wherein the targetingmolecule is not or does not comprise a virus-like particle, ananoparticle, a liposome, a quantum dot, or a combination thereof.

176. The conjugate of any of embodiments 166-175, wherein the first dyethat is a phthalocyanine dye that has a maximum absorption wavelengthfrom or from about 600 nm to about 850 nm.

177. The conjugate of any of embodiments 166-176, wherein the first dyethat is a phthalocyanine dye comprises the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

178. The conjugate of any of embodiments 166-177, wherein the first dyethat is a phthalocyanine dye comprises the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

179. The conjugate of any of embodiments 166-178, wherein the first dyethat is a phthalocyanine dye comprises IRDye 700DX (IR700).

180. The conjugate of any of embodiments 166-179, wherein the secondfluorescent dye exhibits one or more spectral properties selected fromamong fluorescent quantum yield in water, extinction coefficient, Stokesshift, absorption and emission at long wavelength and photostabilitythat is greater compared to the corresponding spectral property of thefirst dye.

181. The conjugate of any of embodiments 166-180, wherein the second dyeis not IR700.

182. The conjugate of any of embodiments 166-181, wherein the second dyeis selected from among hydroxycoumarin, Cascade Blue, Dylight 405,Pacific Orange, Alexa Fluor 430, Fluorescein, Oregon Green, Alexa Fluor488, BODIPY 493, 2.7-Diochlorofluorescien, ATTO 488, Chromeo 488,Dylight 488, HiLyte 488, Alexa Fluor 555, ATTO 550, BODIPY TMR-X, CF555, Chromeo 546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte 555,Dylight 550, BODIPY 564, Alexa Fluor 568, Alexa Fluor 594, Rhodamine,Texas Red, Alexa Fluor 610, Alexa Fluor 633, Dylight 633, Alexa Fluor647, APC, ATTO 655, CF633, CF640R, Chromeo642, Cy5, Dylight 650, AlexaFluor 680, IRDye 680, Alexa Fluor 700, Cy 5.5, ICG, Alexa Fluor 750,Dylight 755, IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800, IRDye 800,Qdot® 525, Qdot® 565, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

183. The conjugate of any of embodiments 166-182, wherein the first dyeis IR700 and the conjugate comprises 1 to 10 or 1 to 5 second dyemolecules per macromolecule.

184. The conjugate of any of embodiments 166-1838, wherein the seconddye exhibits a Stokes shift that is greater than 15 nm, greater than 20nm, greater than 30 nm, greater than 40 nm, greater than 50 nm, greaterthan 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nmor greater than 100 nm.

185. The conjugate of any of embodiments 166-184, wherein the second dyehas a quantum yield in water that is greater than 10%, greater than 15%,greater than 20% or greater than 25%, greater than 30%, greater than40%, greater than 50% or greater.

186. The conjugate of any of embodiments 166-185, wherein the second dyehas an absorption and emission wavelength in the spectrum between orbetween about 650 nm and 950 nm, between or between about 700 nm and1000 nm, between or between about 1000 nm and 1700 nm.

187. The conjugate of any of embodiments 166-186, wherein the first dyeand second dye do not exhibit an overlapping emission and absorptionspectra.

188. The conjugate of any of embodiments 166-187, wherein the second dyeis selected from among ICG, IRDye 680, Alexa Fluor 750, Dylight 755,IRDye 750, Cy7.5, Alexa Fluor 790, Dylight 800 and IRDye 800.

189. The conjugate of any of embodiments 166-188, wherein the second dyeis Alexa Fluor 488, IRDye 680, IRDye 800 or Dylight 755.

190. A composition, comprising the conjugate of any of embodiments159-189. 191. The composition of embodiment 190, further comprising apharmaceutically acceptable excipient.

192. A method of treating a disease or condition in a subjectcomprising:

a) administering to the subject a therapeutically effective amount ofthe conjugate of any of embodiments 159-165 or composition of embodiment190 or embodiment 191, wherein the conjugate binds to a cell present inthe microenvironment of a lesion associated with a disease or condition;and

b) after administering the conjugate, irradiating the lesion at one ormore wavelengths to induce phototoxic activity of the conjugate, therebytreating the disease or condition.

193. A method of treating a disease or condition in a subjectcomprising:

a) administering to the subject a therapeutically effective amount ofthe conjugate of any of embodiments 166-189 or composition of embodiment190 or embodiment 191, wherein the conjugate binds to a cell present inthe microenvironment of a lesion associated with a disease or condition;and

b) after administering the conjugate, irradiating the lesion at one ormore wavelengths to induce phototoxic activity of the first dye of theconjugate and a fluorescent signal of the second dye of the conjugate.

194. The method of embodiment 192 or embodiment 193, comprisingirradiating the lesion at a wavelength that is from or from about 400 toabout 900 nm at a dose of at least 1 J cm⁻² or 1 J/cm of fiber length.

195. The method of embodiment 193 or embodiment 194, comprisingirradiating the lesion with a single wavelength.

196. The method of embodiment 193 or embodiment 195, comprisingirradiating the lesion at two different wavelengths, simultaneously orsequentially, wherein one wavelength induces the phototoxic activity andthe other wavelength induces the fluorescent signal.

197. The method of any of embodiments 192-196, wherein the disease orcondition is a tumor.

198. The method of embodiment 197, comprising irradiating the tumor at awavelength of 660 nm to 740 nm and at a dose of at least 1 J cm⁻²,thereby treating the tumor in the subject.

199. The method of embodiment 197 or embodiment 198, wherein the tumoris a cancer.

200. The method of embodiment 199, wherein the cancer is a cancerlocated at the head and neck, breast, liver, colon, ovary, prostate,pancreas, brain, cervix, bone, skin, eye, bladder, stomach, esophagus,peritoneum, or lung.

201. The method of any of embodiments 197-200, wherein the tumor is asarcoma or carcinoma.

202. The method of embodiment 201, wherein the tumor is a carcinoma thatis a squamous cell carcinoma, basal cell carcinoma or adenocarcinoma.

203. The method of embodiment 202, wherein the tumor is a carcinoma thatis a carcinoma of the bladder, pancreas, colon, ovary, lung, breast,stomach, prostate, cervix, esophagus or head and neck.

204. The method of any of embodiments 1-150 and 192-203, wherein priorto administration of the conjugate the targeting molecule isadministered to the subject.

205. The method of any of embodiments 1-150 and 192-204, wherein thetargeting molecule is administered up to 96 hours prior toadministration of the conjugate.

206. The method of embodiment 204 or embodiment 205, wherein thetargeting molecule is administered at a dose within a range from or fromabout 10 mg/m² to about 500 mg/m².

207. The method of any of embodiments 1-150 and 192-206, wherein thetargeting molecule is an antibody or antigen binding fragment.

208. The method of embodiment 207, wherein the antibody is cetuximab.209. A method of treating a tumor in a subject comprising:

a) administering to a subject a first agent that is an immune modulatingagent;

b) administering to the subject a therapeutically effective amount of aconjugate comprising a phthalocyanine dye linked to a tumor-targetingmacromolecule, wherein the conjugate binds to a tumor; and

c) greater than 12 hours after administering the first agent,irradiating the tumor at a wavelength that renders the conjugatecytotoxic, thereby treating the tumor.

210. The method of embodiment 209, wherein the conjugate binds to a cellsurface molecule of a tumor.

211. The method of embodiment 209 or embodiment 210, wherein theconjugate binds to a cell surface molecule of a tumor cell.

212. The method of any of embodiments 209-211, wherein the first agentis administered at least or at least about 24 hours, 48 hours, 96 hours,one week, two weeks, three weeks or one month prior to irradiating thetumor.

213. The method of any of embodiments 209-212, wherein the first agentis an immune modulating agent that is a demethylating agent thatupregulates expression of a tumor associated antigen (TAA) or is acytokine.

214. The method of any of embodiments 209-213, wherein:

the immune modulating agent is a cytokine that is leukocyte interleukininjection (Multikine); or

the immune modulating agent is a demethylating agent that is5-aza-2′-deoxycytidine.

215. The method of any of embodiments 209-212, wherein the first agentis an immune modulating agent that is an immune checkpoint inhibitor.

216. The method of embodiment 215, wherein the immune checkpointinhibitor specifically binds a molecule selected from among PD-1, PD-L1,PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L,CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

217. The method of any of embodiments 209-216, wherein the immunemodulating agent is a small molecule or a polypeptide.

218. The method of any of embodiments 209-216, wherein the immunemodulating agent is an antibody or an antigen-binding fragment thereof.

219. The method of embodiment 218, wherein the antibody is selected fromamong nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559,MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab,TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469,MEDI4736, MOXR0916, AMP-224, and MSB001078C, or is an antigen-bindingfragment thereof.

220. The method of any of embodiments 209-216, wherein the first agentis an antibody conjugate comprising a phthalocyanine dye linked to anantibody or antigen-binding fragment that is an immune modulating agent.

221. The method of embodiment 220, wherein the immune modulating agentis an immune checkpoint inhibitor.

222. The method of embodiment 2218, embodiment 220 or embodiment 221,wherein the immune modulating agent is an antibody or antibody fragmentthat binds to the surface of a cancer cell.

223. The method of embodiment 222, wherein the immune modulating agentis an antibody or antibody fragment that binds to PD-L1.

224. The method of embodiment 223, wherein the immune modulating agentis an antibody selected from BMS-935559, MEDI4736, MPDL3280A andMSB0010718C, or an antigen-binding fragment thereof.

225. The method of any of embodiments 220-224, wherein step c) ofirradiating the tumor is effected either i) after administration of thefirst agent and after administration of the conjugate or ii) only afteradministration of the conjugate.

226. The method of any of embodiments 220-225, wherein phalocyanine dyeof the first agent and conjugate are the same or different.

227. A method of treating a tumor in a subject comprising:

a) administering to a subject a first agent that is an anti-canceragent;

b) administering to the subject a therapeutically effective amount of aconjugate comprising a phthalocyanine dye linked to a tumor-targetingmacromolecule, wherein the conjugate is binds to a tumor; and

c) greater than 5 minutes after administering the first agent,irradiating the tumor at a wavelength that renders the conjugatecytotoxic, thereby treating the tumor.

228. The method of embodiment 227, wherein the conjugate binds to a cellsurface molecule of a tumor.

229. The method of embodiment 227 or embodiment 228, wherein theconjugate binds to a cell surface molecule of a tumor cell.

230. The method of any of embodiments 227-229, wherein the first agentis administered at least or at least about 15 minutes, 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours or 24 hoursprior to irradiating the tumor.

231. The method of any of embodiments 227-230, wherein the anti-canceragent is selected from among an alkylating agent, a platinum drug, anantimetabolite, an anti-tumor antibiotic, a topoisomerase inhibitor, amitotic inhibitor, a corticosteroid, a proteasome inhibitor, a kinaseinhibitor, a histone-deacetylase inhibitor and an antibody.

232. The method of any of embodiments 227-231, wherein the anti-canceragent is selected from among 5-Fluorouracil/leukovorin, oxaliplatin,irinotecan, ziv-afibercept, capecitabine, cisplatin, paclitaxel,toptecan, carboplatin, gemcitabine, docetaxel, 5-FU, ifosfamide,mitomycin, pemetrexed, vinorelbine, carmustine wager, temozolomide,methotrexate, capacitabine, etoposide, liposomal cytarabine, cytarabine,interferon alpha, vincristine, cyclophosphamide, lomusine, procarbazine,somastostatin, doxorubicin, pegylated liposomal encapsulateddoxorubicin, epirubicin, eribulin, albumin-bound paclitaxel,ixabepilone, cotrimoxazole, taxane, vinblastine, temozolomide,bendamustine, oral etoposide, octreotide, lanredtide, dacarbazine,mesna, eribulin, tamoxifen, toremifene, dactinomycin, enzalutamide,abiraterone acetate, mitoxantrone, cabazitaxel, fluoropyrimidine,oxaliplatin, leucovorin, oxoliplatin and auroropyrimidine.

233. The method of any of embodiments 227-231, wherein the anti-canceragent is selected from bevacizumab, cetuximab, panitumumab, ramucirumab,ipilimumab, rituximab, trastuzumab, ado-trastuzumab emtansine,pertuzumab, nivolumab, lapatinib, dabrafenib, vemurafenib, erlotinib,sunitinib, pazopanib, imatinib, regorafenib, sorafenib, nilotinib,dasantinib, celecoxib, crizotinib, certinib, afatinib, axitinib,bevacizumab, bosutinib, cabozantinib, afatinib, gefitinib, temsirolimus,everolimus, sirolimus, ibrutinib, imatinib, lenvatinib, olaparib,palbociclib, ruxolitinib, trametinib, vandetanib and vismodegib.

234. The method of any of embodiments 20209-233, wherein the conjugateis administered at a dose from or from about 50 mg/m² to 5000 mg/m², 250mg/m² to 2500 mg/m², 750 mg/m² to 1250 mg/m² or 100 mg/m² to 1000 mg/m².

235. The method of any of embodiments 209-234, wherein the conjugate isadministered at a dose of at least 0.01 mg, 0.1 mg, 1 mg, 10 mg, 100 mg,1000 mg, 2000 mg, or 3000 mg.

236. The method of any of embodiments 209-235, wherein the first agentis administered in a dosage range that is from or from about 0.01 mg perkg body weight (mg/kg BW) to about 50 mg/kg BW, about 0.1 mg/kg to about20 mg/kg BW, about 0.1 to about 10 mg/kg BW, about 0.3 to about 10mg/kg, about 0.5 mg/kg to 5 mg/kg or 0.5 mg/kg to 1 mg/kg.

237. The method of any of embodiments 209-236, wherein the conjugate isadministered prior to, simultaneously or subsequently to administrationof the first agent.

238. The method of any of embodiments 209-237, wherein the conjugate andthe first agent are administered prior to irradiating the tumor.

239. The method of any of embodiments 209-226 and embodiment 238,wherein the first agent is an immune modulating agent and the conjugateis administered from or from about 12 hours to 48 hours prior toirradiating the tumor and the immune modulating agent is administeredfrom or from about 12 hours to 1 month prior to irradiating the tumor.

240. The method of any of embodiments 227-239, wherein the first agentis an anti-cancer agent and the conjugate is administered from or fromabout 12 hours to 48 hours prior to irradiating the tumor and theanti-cancer agent is administered from or from about 5 minutes to 24hours prior to irradiating the tumor.

241. The method of any of embodiments 209-240, wherein the first agentand the conjugate are administered by the same route of administration.

242. The method of any of embodiments 209-241, wherein the first agentand the conjugate are administered systemically.

243. The method of any of embodiments 209-242, wherein the first agentand the conjugate are administered intravenously.

244. A method of treating a tumor in a subject comprising:

a) administering to a subject a first conjugate comprising aphthalocyanine dye linked to an antibody or antigen-binding fragmentthat is an immune modulating agent;

b) administering to the subject a therapeutically effective amount of asecond conjugate comprising a phthalocyanine dye linked to atumor-targeting macromolecule, wherein the conjugate binds to a tumor;and

c) irradiating the tumor either i) after administration of the firstconjugate and after administration of the second conjugate or ii) onlyafter administration of the second conjugate, wherein irradiation is ata wavelength that renders the first and second conjugate cytotoxic,thereby treating the tumor.

245. The method of embodiment 244, wherein the second conjugate binds toa cell surface molecule of a tumor.

246. The method of embodiment 244 or 245, wherein the second conjugatebinds to a cell surface molecule of a tumor cell.

247. The method of any of embodiments 244-246, wherein the immunemodulating agent is an immune checkpoint inhibitor.

248. The method of any of embodiments 244-246, wherein the immunemodulating agent is an antibody or antigen binding fragment that bindsto the surface of a cancer cell.

249. The method of any of embodiments 244-248, wherein the immunemodulating agent is an antibody or antibody fragment that binds toPD-L1.

250. The method of embodiment 249, wherein the immune modulating agentis an antibody selected from BMS-935559, MEDI4736, MPDL3280A andMSB0010718C, or an antigen-binding fragment thereof.

251. The method of any of embodiments 244-250, wherein phalocyanine dyeof the first conjugate and the second conjugate are the same ordifferent.

252. The method of any of embodiments 244-251, wherein the firstconjugate is administered prior to administration of the secondconjugate.

253. The method of embodiment 252, wherein the first conjugate isadministered at least 12 hours, at least 24 hours, at least 48 hours, atleast 96 hours, at least one week, at least two weeks, at least threeweeks or at least one month prior to administration of the secondconjugate.

254. The method of any of embodiments 244-253, wherein the methodcomprises a first irradiation of the tumor after administering the firstconjugate and a second irradiation of the tumor after administering thesecond conjugate, wherein each irradiation is performed within 6 to 48hours after administering the respective conjugate.

255. The method of any of embodiments 244-253, wherein:

the method comprises only a single irradiation after administering thesecond conjugate; and

the second conjugate is administered within 6 to 48 hours prior toirradiating the tumor and at least 12 hours after administering thefirst conjugate.

256. The method of any of embodiments 244-255, wherein the firstconjugate and the second conjugate are administered by the same route ofadministration.

257. The method of any of embodiments 244-256, wherein the firstconjugate and the second conjugate are administered systemically.

258. The method of any of embodiments 244-257, wherein the firstconjugate and the second conjugate are administered intravenously.

259. The method of any of embodiments 209-258, wherein thephthalocyanine dye has a maximum absorption wavelength from or fromabout 600 nm to 850 nm.

260. The method of any of embodiments 209-259, wherein thephthalocyanine dye comprises a linker comprising a reactive group forattachment of the dye to the tumor-targeting macromolecule.

261. The method of embodiment 260, wherein the phthalocyanine dyecomprises the formula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the tumor-targetingmacromolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

262. The method of embodiment 260 or embodiment 261, wherein thephthalocyanine dye comprises the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

263. The method of any of embodiments 209-262, wherein thephthalocyanine dye comprises IRDye 700DX (IR700).

264. The method of any of embodiments 220-226, wherein phalocyanine dyeof the first agent and the conjugate are the same and each is IR700.

265. The method of any of embodiments 244-258, wherein phalocyanine dyeof the first conjugate and the second conjugate are the same and each isIR700.

266. The method of any of embodiments 209-265, wherein thetumor-targeting macromolecule comprises a molecule or biomoleculeselected from a protein, a glycoprotein, an antibody, an antibodyfragment, an antigen, an antigen binding fragment, a peptide, apolypeptide, a small molecule, a polymeric synthetic molecule, apolymeric nanoparticle, a liposome, an enzyme substrate, a hormone, aneurotransmitter, a cell metabolite, a viral particle, a viral capsid, aviral nanoparticle, a bacterial particle, a marker, a cell, a hapten, anavidin, a streptavidin, biotin, a carbohydrate, an oligosaccharide, apolysaccharide, a nucleic acid, a deoxy nucleic acid, a fragment of DNA,a fragment of RNA, nucleotide triphosphates, acyclo terminatortriphosphates, and PNA.

267. The method of embodiment 266, wherein the tumor targetingmacromolecule binds to a cell surface molecule expressed in tumors orcancer cells.

268. The method of embodiment 267, wherein cell surface moleculecomprises an antigen, a polypeptide, a lipid, or a carbohydrate or acombination of these molecules.

269. The method of embodiment 267 or embodiment 268, wherein the cellsurface molecule is selected from among ACTHR, endothelial cell Anxa-1,aminopetidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin,alpha-5-beta-5 integrin, alpha-fetoprotein (AFP), ANPA, ANPB, APA, APN,APP, 1AR, 2AR, AT1, B1, B2, BAGE1, BAGE2, B-cell receptor BB1, BB2, BB4,calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10,CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52,CD56, CD68, CD90, CEA (CarcinoEmbryonic Antigen), CGRP, chemokinereceptors, cell-surface annexin-1, cell-surface plectin-1, Cripto-1,CRLR, CXCR2, CXCR4, DCC, E2 glycoprotein, EGFR, EGFRvIII, EMR1,Endosialin, EP2, EP4, ET receptors, Fibronectin, Fibronectin ED-B, FGFR,frizzled receptors, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1receptor, G-protein coupled receptors of the Family A (Rhodopsin-like),G-protein coupled receptors of the Family B (Secretin receptor-like)like), G-protein coupled receptors of the Family C (MetabotropicGlutamate Receptor-like), GD2, GP100, GP120, hemagglutinin, Heparinsulfates, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7 antigens,hTERT, IL11-R, IL-13R, ITGAM, Kalikrien-9, Lewis Y, LH receptor, LHRH-R,LPA1, MAC-1, MAGE 1, MAGE 2, MAGE 3, MAGE 4, MART 1, MC1R, Mesothelin,MUC1, MUC16, Neu (cell-surface Nucleolin), Neprilysin, Neuropilin-1,Neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, Notch-1, NY-ESO-1, OT-R, mutantp53, p97 melanoma antigen, NTR2, NTR3, p32 (p32/gC1q-R/HABP1), p75,PAC1, PAR1, Patched (PTCH), PDGFR, PDFG receptors, PDT, Protease-cleavedcollagen IV, proteinase 3, prohibitin, protein tyrosine kinase 7, PSA,PSMA, purinergic P2X family (e.g., P2X1-5), mutant Ras, RAMP1, RAMP2,RAMP3 patched, RET receptor, plexins, smoothened, sst1, sst2A, sst2B,sst3, sst4, sst5, substance P, TEMs, T-cell CD3 Receptor, TAG72, TGFBR1,TGFBR2, Tie-1, Tie-2, Trk-A, Trk-B, Trk-C, TR1, TRPA, TRPC, TRPV, TRPM,TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPML1-3),TSH receptor, VEGF receptors (VEGFR1 or Flt-1, VEGFR2 or FLK-1/KDR, andVEGF-3 or FLT-4), voltage-gated ion channels, VPAC1, VPAC2, Wilms tumor1, Y1, Y2, Y4, and Y5.

270. The method of any of embodiments 267-269 wherein the cell surfacemolecule is selected from among HER1/EGFR, HER2/ERBB2, CD20, CD25(IL-2Ra receptor), CD33, CD52, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6,cancer antigen 125 (CA125), alpha-fetoprotein (AFP), Lewis Y, TAG72,Caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor,vascular endothelial growth factor (VEGF), CD30, EpCAM, gpA33, mucins,CAIX, PSMA, folate-binding protein, gangliosides (such as GD2, GD3, andGM2), VEGF receptor (VEGFR), integrin αVβ3, integrin α5β1, ERBB3, MET,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex,CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 (3, HLA-DR antigen, IgE, MUC-1,nuC242, PEM antigen, metalloproteinases, Ephrin receptor, Ephrinligands, HGF receptor, CXCR4, CXCR4, Bombesin receptor, and SK-1antigen.

271. The method of any of embodiments 209-270, wherein thetumor-targeting macromolecule is an antibody or an antibody fragment.

272. The method of embodiment 271, wherein the antibody is selected fromcetuximab, panitumumab, zalutumumab, nimotuzumab, Tositumomab (Bexxar®),Rituximab (Rituxan, Mabthera), Ibritumomab tiuxetan (Zevalin),Daclizumab (Zenapax), Gemtuzumab (Mylotarg), Alemtuzumab, CEA-scan Fabfragment, OC125 monoclonal antibody, ab75705, B72.3, Bevacizumab(Avastin®), and Basiliximab, nivolumab, pembrolizumab, pidilizumab,MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31,BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40,CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C,or is an antibody-binding fragment thereof.

273. The method of embodiment 272, wherein the antibody is selected fromcetuximab, panitumumab and trastuzumab.

274. The method of any of embodiments 209-273, wherein the conjugate isselected from cetuximab-IR700, panitumumab-IR700 and trastuzumab-IR700.

275. The method of any of embodiments 209-274, wherein thetumor-targeting macromolecule is a tissue-specific homing peptide.

276. The method of embodiment 275, wherein the homing polypeptidecomprises the sequence of amino acids set forth in any of SEQ ID NOS:1-52.

277. The method of any of embodiments 209-276, wherein thetumor-targeting macromolecule is an RGD polypeptide, an iRGDpolypeptide, a Lyp-1 polypeptide, a cripto-1 binding polypeptide, asomatostatin receptor binding polypeptide, a prohibitin bindingpolypeptide, a NGR polypeptide, or an iNGR polypeptide.

278. The method of any of embodiments 209-277, wherein thetumor-targeting macromolecule is selected from among adrenocorticotropichormone (ACTH), angiotensin II, atrial natriuretic factor (ANF),bombesin, bradykinin, brain derived neurotropihic factor (BDNF), bonemorphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6),bone morphogenetic protein 7 (BMP-7), calcitonin, cardiotrophin 1(BMP-2), CD22, CD40, cholecystokinin (CCK), ciliary neurotrophic factor(CNTF), CCL1-CCL28, CXCL1-CXCL17, XCL1, XCL2, CX3CL1, cripto 1 bindingpeptide, vascular endothelial cell growth factor (VEGF), epidermalgrowth factor (EGF), endothelin 1, endothelin 1/3, FAS-ligand,fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2),fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5),fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7),fibroblast growth factor 1 (FGF-10), Flt-3, gastrin, gastrin releasingpeptide (GRP), granulocyte colony-stimulating factor (G-CSF),granulocyte macrophage stimulating factor (GM-CSF), glucagon likepeptide (GLP-1), hepatocyte growth factor (HGF), interferon alpha(IFN-α), interferon beta (IFN-b), interferon gamma (IFNg), insulin-likegrowth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2),interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9),interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12),interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17),interleukin 19 (IL-19), luteinizing hormone (LH), luteinizing-releasinghormone (LHRH), macrophage colony-stimulating factor (M-CSF), monocytechemotactic protein 1 (MCP-1), macrophage inflammatory protein 3a(MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growthfactor (NGF), neuromedin B, neurotrophin 3 (NT-3), neurotrophin 4(NT-4), neurotensin, neuropeptide Y, oxytocin, pituitary adenylatecyclase activating peptide (PACAP), platelet derived growth factor AA(PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derivedgrowth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC),platelet derived growth factor DD (PDGF-DD), netrin-1 (NTN1), netrin-2(NTN2), netrin-4 (NTN4), netrin-G1 (NTNG1) and netrin-G2 (NTNG2), ephrinA1 (EFNA1), ephrin A2 (EFNA2), ephrin A3 (EFNA3), ephrin A4 (EFNA4),ephrin A5 (EFNAS), semaphorin 3A (SEMA3A), semaphorin 3B (SEMA3B),semaphorin 3C (SEMA3C), semaphorin 3D (SEMA3D), semaphorin 3F (SEMA3F),semaphorin 3G (SEMA3G), semaphorin 4A (SEMA4A), semaphorin 4B (SEMA4B),semaphorin 4C (SEMA4C), semaphorin 4D (SEMA4D), semaphorin 4F (SEMA4F),semaphorin 4G (SEMA4G), semaphorin 5A (SEMA5A), semaphorin 5B (SEMA5B),semaphorin 6A (SEMA6A), semaphorin 6B (SEMA6B), semaphorin 6D (SEMA6D),semaphorin 7A (SEMA7A), SLIT1, SLIT2, SLIT3, SLIT and NTRK-like family,member 1 (SLITRK1), SLIT and NTRK-like family, member 2 (SLITRK2), SLITand NTRK-like family, member 3 (SLITRK3), SLIT and NTRK-like family,member 4 (SLITRK4), SLIT and NTRK-like family, member 5 (SLITRK5), SLITand NTRK-like family, member 6 (SLITRK6), prostaglandin E2 (PGE2),RANTES, Somatostatin-14, Somatostatin-28, stem cell factor (SCF),stromal cell derived factor 1 (SDF-1), substance P, thyroid stimulatinghormone (TSH), transforming growth factor alpha (TGF-α), transforminggrowth factor beta (TGF-b), tumor necrosis factor alpha (TNF-α),thrombin, vasoactive intestinal peptide (VIP), Wnt1, Wnt2, Wnt2b/13,Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a,Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, or Wnt16, Sonichedgehog, Desert hedgehog, and Indian hedgehog.

279. The method of any of embodiments 209-278, wherein the conjugatecomprises from or from about 1 to 1000 phthalocyanine dye molecules permacromolecule.

280. The method of any of embodiments 209-279, wherein the conjugatecomprises 1 to 100, 1 to 10 or 2 to 5 phthalocyanine dye molecules permacromolecule.

281. The method of any of embodiments 209-279, wherein the macromoleculeis a nanoparticle and the conjugate comprises 100 to 1000 phthalocyaninedye molecules per macromolecule.

282. The method of any of embodiments 209-281, wherein the tumor is acancer.

283. The method of any of embodiments 209-282, wherein the cancer is acancer located at the head and neck, breast, liver, colon, ovary,prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach,esophagus, peritoneum, or lung.

284. The method of any of embodiments 209-282, wherein the cancer is acancer of the blood.

285. The method of any of embodiments 209-284, wherein the tumor isirradiated at a wavelength of 600 to 850 nm at a dose of at least 1 Jcm⁻².

286. The method of any of embodiments 209-285, wherein the tumor isirradiated at a wavelength of 690 nm±50 nm.

287. The method of any of embodiments 209-286, wherein the tumor isirradiated at a wavelength of or about 690±20 nm.

288. A conjugate, comprising a phthalocyanine dye linked to an antibodyor antigen-binding fragment that is an immune modulating agent.

289. The conjugate of embodiment 288, wherein the immune modulatingagent is an immune checkpoint inhibitor.

290. The conjugate of embodiment 288 or embodiment 289, wherein theimmune modulating agent is an antibody or antigen binding fragment thatbinds to the surface of a tumor, tumor cell or cancer cell.

291. The conjugate of any of embodiments 288-290, wherein the immunemodulating agent is an antibody or antibody fragment that binds toPD-L1.

292. The conjugate of embodiment 291, wherein the immune modulatingagent is an antibody selected from BMS-935559, MEDI4736, MPDL3280A andMSB0010718C, or an antigen-binding fragment thereof.

293. A composition, comprising the conjugate of any of embodiments288-292.

294. A pharmaceutical composition, comprising the conjugate of any ofembodiments 288-293 and a pharmaceutically acceptable carrier.

295. A combination, comprising:

a first composition comprising an immune modulating agent, with theproviso that the immune modulating agent is not 5-aza-2′-deoxycytidineor an anti-CTLA4 antibody; and

a second composition comprising a conjugate comprising a phthalocyaninedye linked to a tumor-targeting macro molecule, wherein the conjugatebinds to a tumor.

296. The combination of embodiment 295, wherein the conjugate binds to acell surface molecule of a tumor.

297. The combination of embodiment 295 or embodiment 296, wherein theconjugate binds to a cell surface molecule of a tumor cell.

298. The combination of any of embodiments 295-297, wherein the immunemodulating agent is an immune checkpoint inhibitor.

299. The combination of embodiment 298, wherein the immune checkpointinhibitor specifically binds a molecule selected from among PD-1, PD-L1,PD-L2, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2,B7-H3, B7-H4, BTLA, HVEM, CD28 and VISTA.

300. The combination of any of embodiments 295-299, wherein the immunemodulating agent is a small molecule or a polypeptide.

301. The combination of any of embodiments 295-300, wherein the immunemodulating agent is an antibody or an antigen-binding fragment thereof.

302. The combination of embodiment 296, wherein the antibody is selectedfrom among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559,MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab,TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469,MEDI4736, MOXR0916, AMP-224, and MSB001078C, or is an antigen-bindingfragment thereof.

303. The conjugate of any of embodiments 288-292 or combination of anyof embodiments 295-302, wherein the phthalocyanine dye comprises theformula:

wherein:

L is a linker;

Q is a reactive group for attachment of the dye to the targetingmolecule;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group;

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²² and R²³ are eachindependently selected from hydrogen, halogen, optionally substitutedalkylthio, optionally substituted alkylamino and optionally substitutedalkoxy; and

X² and X³ are each independently C₁-C₁₀ alkylene, optionally interruptedby a heteroatom.

304. The conjugate or combination of embodiment 288-292 or 295-303,wherein the phthalocyanine dye comprises the formula:

wherein:

X¹ and X⁴ are each independently a C₁-C₁₀ alkylene optionallyinterrupted by a heteroatom;

R², R³, R⁷, and R⁸ are each independently selected from optionallysubstituted alkyl and optionally substituted aryl;

R⁴, R⁵, R⁶, R⁹, R¹⁰, and R¹¹ are each independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkanoyl,optionally substituted alkoxycarbonyl, optionally substitutedalkylcarbamoyl, and a chelating ligand, wherein at least one of R⁴, R⁵,R⁶, R⁹, R¹⁰, and R¹¹ comprises a water soluble group; and

R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are each independently selected from hydrogen,halogen, optionally substituted alkylthio, optionally substitutedalkylamino and optionally substituted alkoxy.

305. The conjugate or combination of any of embodiments 288-292 or295-2304 wherein the phthalocyanine dye comprises IRDye 700DX (IR700).

306. A kit, comprising the conjugate, composition or combination of anyof embodiments 288-305 and, optionally, instructions for use.

307. An article of manufacture, comprising the conjugate, composition orcombination of any of embodiments 288-305.

308. The article of manufacture of embodiment 307 that is a container.

309. The article of manufacture of embodiment 308, wherein the containerprotects from transmission of light having a wavelength from or fromabout 500 to 725 or 650 to 725.

3010. The article of manufacture of embodiment 309, wherein thepercentage of light transmission through the container is less than 50%,less than 40%, less than 30%, less than 20%, less than 10% or less than5%.

311. The article of manufacture of any of embodiments 308-310, whereinthe container is green, amber, translucent, opaque, or is wrapped in anopaque foil.

312. A method of treating a tumor in a subject comprising:

a) administering to a subject a conjugate of any of embodiments 288-292,composition of embodiment 293 or embodiment 294 or combination of any ofembodiments 295-302; and

c) irradiating the tumor at a wavelength that renders the conjugatecytotoxic, thereby treating the tumor

313. The method of embodiment 312, wherein the tumor is a cancer.

314. The method of embodiment 312 or embodiment 313, wherein the canceris a cancer located at the head and neck, breast, liver, colon, ovary,prostate, pancreas, brain, cervix, bone, skin, eye, bladder, stomach,esophagus, peritoneum, or lung.

315. The method of any of embodiments 312-314, wherein the cancer is acancer of the blood.

316. The method of any of embodiments 312-315, wherein the tumor isirradiated at a wavelength of 600 to 850 nm at a dose of at least 1 Jcm⁻².

317. The method of any of embodiments 312-316, wherein the tumor isirradiated at a wavelength of 690 nm±50 nm.

318. The method of any of embodiments 312-317, wherein the tumor isirradiated at a wavelength of or about 690±20 nm.

VII. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Generation of Cetuximab-IRDye 700DX Conjugate

This example describes a method for preparing a conjugate containingIRDye 700DX (IR700) linked to cetuximab to produce cetuximab-IRDye 700DX(cetuximab-IR700). The provided methods are exemplary and similarmethods may be employed to conjugate another targeting molecule to IRDye700Dx. The methods were performed to limit exposure of the dye andconjugate to light due to the photosensitivity of the dye, whichincluded the use of low levels of green light having a wavelength from425 to 575 nm and an intensity of less than 200 Lux in the manufacturingfacility.

A. Preparation of Buffers

Buffers were prepared using Highly Purified Water (HPW) or Water forInjection (WFI) and were filtered through a 0.22 μm filter prior tostorage at ambient temperature. Tables 2-4 show in-process controls andtests for prepared buffers: conjugation buffer (100 mM sodium phosphate,pH 8.65), quenching buffer (1.0 M glycine, pH 9) and final phosphatebuffered saline (PBS) formulation buffer: (5.60 mM Na₂HPO₄, 1.058KH₂PO₄, 154 mM NaCl, pH 7.1), respectively.

TABLE 2 Preparation of Conjugation Buffer (100 mM sodium phosphate, pH8.65) In-process Controls and Tests Specification or Range Mixing time≥30 min pH 8.5-8.8 Conductivity 11.7-14.1 mS/cm Filter integrity testingPass Endotoxin ≤1.5 EU/mL

TABLE 3 Preparation of Quenching Buffer (1.0M glycine, pH 9) In-processControls and Tests Specification or Range Mixing time ≥30 min pH 8.9-9.1Conductivity 5-11 mS/cm Filter integrity testing Pass Endotoxin ≤1.5EU/mL

TABLE 4 Buffer release test for 1x PBS Tests Specification or RangeAppearance Clear solution pH 7.0-7.2 Osmolality 280-330 mOsm/kgSterility No growth Cytotoxicity Non-toxic

B. Preparation of Dye and Cetuximab

1. Cetuximab Preparation

Prior to conjugation, Cetuximab vials (Myoderm USA, Norristown, Pa.)were sprayed with sterile isopropyl alcohol and placed in a Laminar FlowHood. A total of 423 vials were used to prepare drug substance. Thevials were de-crimped using an electronic decrimper, the stoppers wereremoved with autoclaved forceps, and the contents were poured intosterile 2 L PETG bottles. The bottles were capped when filled. TheCetuximab was then filtered through a 0.22 μm filter and pooled into a50 L HyQtainer. Pooled, filtered Cetuximab was stored at 2-8° C.

A concentration and buffer exchange step was then performed byultrafiltration/diafiltration (UF/DF). Cleaning of the UF/DF device wasperformed prior to use. The storage solution was drained and themembrane flushed with at least 20 L of HPW. The unit was flushed with0.1 M NaOH for 30-40 min and then flushed with HPW. The pH of therinsate was confirmed. The system was equilibrated with 100 mM sodiumphosphate, pH 8.65 buffer. Permeate and retentate effluent pH andconductivity were confirmed prior to use. Endotoxin testing was alsoperformed; the system was used within 48 hours of endotoxin testing.

Prior to UF/DF operations, the pooled, filtered Cetuximab was warmed byplacing it in an incubator at 25° C. for 120-150 min. The material wasfirst concentrated to a target of 5 mg/mL and then diafiltered into 100mM sodium phosphate, pH 8.65 buffer. Diafiltration was performed untilthe permeate pH and conductivity targets were met. The system wasflushed with buffer and the flush was added to the diafilteredretentate. UF/DF system pressures were monitored and recorded during theoperation as described in Table 5.

The diafiltered Cetuximab product concentration was determined and thendiluted to a target concentration of 2 mg/mL (1.8-2.4 mg/mL) using 100mM sodium phosphate, pH 8.65 buffer. The product was asepticallyfiltered through a 0.22 μm filter and split into two autoclavedproduct-dedicated 40 L carboys containing stir bars andforward-processed directly into the conjugation operation. The weight ofCetuximab in each carboy was determined.

TABLE 5 In-process controls and tests for Cetuximab processingIn-process Controls and Tests Specification or Range Cetuximab poolingFilter integrity test (0.22 μm) (after pooling) Pass Proteinconcentration after pooling Report results TFF Unit Preparation TFFparts 1.0M NaOH contact time ≥60 min pH of TFF rinsed parts <7 TOC ofUF/DF rinsed parts <1000 ppb HPW volume rinse with membrane >20 L UF/DFIntegrity testing prior to use Air displacement <90 mL/min 0.1M NaOHflush time 30-40 min UF/DF permeate and retentate pH after <7 HPWrinsing TFF Equilibration UF/DF permeate and retentate effluent pH8.5-8.8 UF/DF permeate and retentate effluent 11.7-14.1 mS/cmconductivity UF/DF permeate and retentate effluent ≤0.134 EU/mLendotoxin Cetuximab Diafiltration Pooled, filtered Cetuximab incubation25° C. temperature Pooled, filtered Cetuximab incubation time 120-150min Feed inlet pressure during concentration <25 psi Retentate outletpressure during concentration 10-12 psi Retentate pressure duringdiafiltration 10-12 psi UF/DF system pressure during diafiltration <32psi UF/DF permeate pH after diafiltration 8.5-8.8 UF/DF permeateconductivity after 11.7-14.1 mS/cm diafiltration Cetuximab concentration(A₂₈₀) after 4.5 mg/mL diafiltration Cetuximab concentration (A₂₈₀)after 1.8-2.4 mg/mL dilution

2. Dye Preparation

Prior to conjugation, IRDye 700DX NHS Ester (dye; Cat. No. 929-70011;Li-COR, Lincoln, Nebr.) was prepared by dissolving it to a concentrationof 10 mg/mL in anhydrous DMSO. The steps were performed under greenlight (e.g., wavelength from 425 to 575 nm and an intensity of less than200 Lux) to protect the dye from the wavelengths of light that arestrongly absorbed by the dye.

C. Conjugation

The conjugation and quenching steps were performed in the 2×40 L carboys(wrapped in aluminum foil for light protection) containing diafilteredCetuximab. The steps were performed at room temperature under greenlight (e.g., wavelength from 425 to 575 nm and an intensity of less than200 Lux) to protect the conjugate from photo-degradation.

For the conjugation reaction, the appropriate amount of IRDye 700DX NHSester in DMSO was calculated (based on the weight of Cetuximab in eachcarboy, typically from 80-120 g) to achieve a final molar ratio of 4:1(IRDye 700DX NHS ester: Cetuximab). Process development studies havedetermined that this ratio, in conjugation with the targeted conjugationincubation time, should incorporate 2-3 dye residues per Cetuximabmolecule. The calculated amount of the IRDye 700DX NHS ester was addedto the carboys containing Cetuximab and mixed on a stir plate for 10-15min. The conjugation reaction then proceeded for 120 min by placing thecarboys in a 25° C. incubator.

The conjugation reaction was quenched by the addition of 1 M glycine toa final concentration of 4.2 mM and mixing for 10-12 min. The carboyswere incubated for an additional 20-25 min in the 25° C. incubator.Table 6 displays in-process controls and tests for the conjugation andquenching steps.

TABLE 6 In-process controls and tests for conjugation and quenchingsteps In-process Controls and Tests Specification or Range Conjugationstep mixing time 10-15 min Conjugation step incubation time 120(115-125) min Conjugation step incubation temperature 25 (23-27) ° C.Quenching step mixing time 10-12 min Quenching step incubation time20-25 min Quenching step incubation temperature 25 (23-27) ° C.

A final UF/DF step was performed to exchange the conjugated product intothe final PBS formulation buffer. Cleaning of the UF/DF system wasperformed prior to use. The unit was cleaned and parts were soaked in1.0 M NaOH and then rinsed with HPW. The system was equilibrated withPBS, pH 7.1 until the permeate was within specifications. Permeate andretentate were tested for endotoxin.

The quenched conjugate was transferred to the UF/DF system and was firstconcentrated to 8-10 L followed by diafiltration with 8-12 diavolumes ofPBS in order to exchange the product into the final formulation buffer.The pH and conductivity were confirmed. The system was flushed withbuffer and the flush was added to the final product. The proteinconcentration was determined and if needed, further dilution with PBSwas performed to reach a final target product concentration of 2.0 mg/mL(1.8-2.1 mg/mL).

A final filtration through a 0.22 μm filter was performed and theCetuximab-IRDye 700DX conjugate was stored in the dark at 2-8° C. in a50 L HyQtainer covered with aluminum foil to protect the contents fromlight. The steps were performed at room temperature under green light toprotect the Cetuximab-IRDye 700DX conjugate. Table 7 displays in-processcontrols and tests for the final UF/DF, filtration, and storage. In somecases, dilution was required.

TABLE 7 In-process controls and tests for final UF/DF, filtration, andstorage In-process Controls and Tests Specification or Range TFF UnitPreparation 0.1M NaOH flush time 30-40 min HPW rinse volume ≥20 L TFFEquilibration pH of permeate after equilibration 7.0-7.2 UF/DF permeateand retentate ≤0.134 EU/mL effluent endotoxin Cetuximab-IRDye 700DXConjugate Diafiltration pH of permeate after diafiltration 7.0-7.2Conductivity of permeate after 11-16 mS/cm diafiltration Targetconjugate protein 1.8-2.1 mg/mL concentration (SEC-HPLC) afterdiafiltration Filter integrity test Pass

After preparing the conjugated material, the sample was submitted forSEC-HPLC to determine concentration, dye to antibody ratio (DAR),identity and purity. Other tests for appearance, pH, bioburden, andendotoxin level also were performed. Table 8 shows the results of thesetests for an exemplary batch product with reference to generalacceptance criterion for the drug substance.

TABLE 8 Drug Substance Specifications Pass/ Test Acceptance CriterionResult Fail Appearance Green to blue liquid Conforms Pass May containvisible particulates Bioburden <1 CFU/mL 0 CFU/mL Pass Endotoxin (LAL)≤0.067 EU/mg <0.06 EU/mg Pass pH 7.1 ± 0.5 7.1 Pass Concentration by 1.8to 2.1 mg/mL 2.0 mg/mL Pass SEC-HPLC DAR by SEC- 1.5 to 4.0 2.9 PassHPLC (A690/ A280 with dye correction) Identity by SEC- Relativeretention Relative retention Pass HPLC (A690) time of monomer time ofmonomer peak: peak: 0.99 of 0.90 to 1.10 of Reference Standard ReferenceStandard monomer peak monomer peak Purity by SEC- Monomer ≥ 92.0%Monomer 100.0% Pass HPLC (A690) HMW ≤  5.0% HMW  0.0% LMW ≤  5.0% LMW 0.0% Free Dye: ≤   3% Free Dye:    0%

Example 2: Pharmacokinetics and Therapeutic Efficacy of Cetuximab-IRDye700DX Conjugate

This Example describes the interim results of a clinical study (Phases 1and 2) assessing safety and efficacy in head and neck cancer patientstreated with a single or multiple administration of cetuximab-IRDye700DX conjugate followed, by irradiation to induce photoimmunotherapy(PIT). Pharmacokinetic parameters and tumor response in human patientsafter single dose administration of cetuximab-IRDye 700DX conjugate weredetermined to evaluate safety and efficacy of the therapy.

1. Methods

Nine (9) patients with squamous carcinoma of head and neck entered adose escalation clinical trial. The patients were divided into three (3)dose cohorts, as listed in Table 8A below. Each cohort included three(3) patients. All patients enrolled in the trial had recurrentprogressive cancers that had failed multiple rounds of commerciallyavailable treatments, some of which had failed previous treatment withthe antibody Cetuximab. The study included both patients with HPVpositive and negative tumors, and patients with P16 positive andnegative tumors.

To ensure a safe initial dose based on ICH guidance for first in humanadministration of experimental therapeutics, a starting dose of 4.3mg/kg (160 mg/m²) was used in the dose escalation study. The startingdose was below the threshold of 6-fold below the Highest Non-SeverelyToxic Dose (HNSTD).

TABLE 8A Dose Cohorts for Phase I Clinical Study of Cetuximab-IRDye700DX No. of Human Clinical Human Clinical Cohort Patients Dose (mg/kg)Dose (mg/m²) 1 3 4.0 160 2 3 8.0 320 3 3 16.0 640

Intravenous (IV) bags containing the conjugate were prepared from vialscontaining 50 mL of a 2 mg/mL solution of cetuximab-IRDye 700DXconjugate produced as described in Example 1. As described in Example 1,the vials were packaged in a single carton and then in an opaque pouchprior to use. The handling of cetuximab-IRDye 700DX conjugate and itsadministration by infusion were performed in a darkened room with lessthan 400 lux of fluorescent light. No tungsten lighting was ever usedduring the preparation of the of the infusion bags. Any windows in theroom were covered with shades so that the cetuximab-IRDye 700DXconjugate was never directly or indirectly exposed to sunlight.

In a biosafety cabinet or hood with the light switched-off so that theconjugate was exposed to an intensity of light of no more than 200 lux(equivalent to 60 Watt light bulb or 15 Watt fluorescent room light),each vial was removed from the opaque couch and then from the carton.The packaging of each vial containing the conjugate was opened and thecontents of that vial were placed into a sterile IV bag until thedesired dose of conjugate for infusion was achieved. Each vial wasopened separately and placed into the IV bag so as to reduce theexposure of the drug product to ambient room light. The process wasperformed in less than 15 min. The IV bag was covered at all times by anopaque sleeve to protect the conjugate from light exposure. Afterpreparation, the IV bag was stored at 2-8° C. for up to 1 hour.

Prior to conjugate administration, the subjects were pretreated with 100mg of Erbitux® (non-conjugated cetuximab) administered by IV infusionover 30 minutes as a screening step for evaluating possible infusionreactions. During the infusion, the subject was evaluated for possibleinfusion reactions to Erbitux® of Grade 3 or greater, which did notoccur in any of the treated patients. After the 100 mg Erbitux®infusion, but just prior to cetuximab-IRDye 700DX conjugate infusion,the subjects were pre-treated with 50 mg of anti-histaminic Benadryl(Diphenhydramine) and 10 mg of the steroid Decadron (Dexamethasone) byIV administration to limit the risk of hypersensitivity tocetuximab-IRDye 700DX conjugate infusion.

The patients were intravenously administered with a single dose of thecetuximab-IRDye 700DX conjugate at the clinical doses set forth above inTable 8A. The conjugate was administered via IV infusion over 2 hours onDay 1. The intravenous (IV) infusion bag was covered during theadministration by an opaque sleeve to protect the conjugate from lightexposure. Any light exposure was limited to less than 5 minutes. If theflow of the cetuximab-IRDye 700DX conjugate during the infusion from theIV infusion bag was stopped for more than 5 minutes, the tubing andfilter were protected from light exposure using an opaque cover, such asaluminum foil.

To induce photoimmunotherapy (PIT), one light application with a lighthaving a wavelength of 690 nm was performed at 24 hours±3 hours (Day 2)post conjugate administration. 690 nm light was administered to thetumor via 400 micron glass fiber microlens forward firing fibers forsurface illumination of tumors that were less than 10 mm thick, or viacylindrical diffuser fibers placed into the tumor for tumors that weregreater than 10 mm thick or for subcutaneous tumors. Light treatment wasfixed at a low fluence of 50 J/cm² for superficial illumination or 100J/cm fiber length for interstitial illumination (at a fluence rate of150 mW/cm² for superficial illumination and 400 mW/cm fiber length forinterstitial illumination).

For microlens surface light treatment, normal tissue located 0.5-1.0 cmaround the periphery of the tumor was also included in the lighttreatment field to reach microscopic infiltrating disease at the marginof the tumor. All other normal tissue were covered with surgical towels,moist surgical sponges or cottonoids to prevent reflected 690 nm lightfrom potentially activating the cetuximab-IRDye 700DX conjugate innormal cutaneous tissues, to reduce the risk of edema, ulceration ornecrosis of normal tissue. In the oral cavity, pharynx and larynx, alltissues not to be treated were covered with moist surgical sponges orcottonoids to prevent reflected light from activating cetuximab-IRDye700DX conjugate that may be present in the mucosa. If the larynx wastreated, the entire laryngeal airway was protected from light exposureusing moist surgical sponges. The microlens fiber was connected to thelaser console according to the manufacturer's instructions. Lighting inthe treatment room or operating room were standard overhead roomlighting, with no high intensity surgical lights turned on in the room.At all times, Class IV Medical Laser precautions were observed.

For cylindrical diffuser implantation directly into tumors, Besttransparent 17 Fr brachytherapy catheters were placed into the tumor1.8+/−0.2 cm apart to uniformly illuminate the entire tumor volume plusat least 0.5 cm margin of normal tissue around the tumor. Standardtechniques were used to place brachytherapy catheters, includingultrasound (US) or computerized tomography (CT) guidance based oninterventional radiologic methods. In some instances, a brachytherapygrid was employed to place the fibers 1.8 cm apart and parallel to eachother in the treatment field. Positioning of the catheters was confirmedby lateral X-ray, US or CT. The cylindrical diffuser fibers were thenconnected to the 690 nm laser console, according to the manufacturer'sinstructions. After ensuring proper light output of each cylindricaldiffuser, the cylindrical diffuser fibers of previously determinedlength (1.0, 2.0, 3.0 or 4.0 cm length) depending on tumor diameter,were placed down the catheter up to its tip and secured in place to thecatheter externally with surgical tape. Lighting in the treatment roomor operating room were standard overhead room lighting, with no highintensity surgical lights turned on in the room. At all times, Class IVMedical Laser precautions were observed.

None of the patients showed adverse effects to the infusion and did notreport any pain. No skin photosensitivity to ambient light was detected.

2. Response and Pharmacokinetics

Patients with head and neck cancer patients treated with a singleadministration of cetuximab-IRDye 700DX conjugate followed byirradiation to induce photoimmunotherapy (PIT) were assessed for tumorresponse. The tumor response was evaluated according to the RECIST(Response Evaluation Criteria In Solid Tumors) criteria as outlined inthe revised version 1.1 guidelines (RECIST 1.1, see Eisenhauer et al.(2009) European Journal of Cancer, 45:228-247). A response wasdetermined to be a “complete response” (CR) if there was a disappearanceof all target lesions, and any pathological lymph nodes (whether targetor non-target) were reduced in short axis to <10 mm. A response wasdetermined to be a “partial response” (PR) if there was at least a 30%decrease in the sum of diameter of target lesions (e.g., at least 30%reduction in tumor growth), taking as reference the baseline sumdiameters of the target lesions prior to the treatment. The “objectiveresponse rate” (ORR) is the percentage of subjects in which a CR or PRresponse was observed.

The tumor response was observed as early as four days with evidence oftumor necrosis. All patients in all three cohorts showed a partial orcomplete response to the PIT treatment.

To examine exposure of cetuximab in the blood from the loweradministered single dose of cetuximab-IRDye 700Dx conjugate, bloodsamples from each patient were collected and subjected topharmacokinetic analyses. The exposure of Cetuximab-IRDye 700DX in thehuman subjects was determined by bioanalytical studies using GoodLaboratory Practice validated methods that specifically detectCetuximab-IRDye 700DX in human serum. Serum concentration versus timewas analyzed by a non-compartment model with linear elimination. Thearea under the curve (AUC) at 24 hours (AUC 0-24) described the totalamount of drug exposure in human blood from the infusion to the time ofpharmacological activity at 24 h post drug infusion that is when thedrug is activated at the tumor to induce cancer killing in a localizedfashion.

A summary of the pharmacokinetic variables for the cetuximab-IRDye 700DXconjugate in Cohort 1 (4 mg/kg; 160 mg/m² dose), Cohort 2 (8 mg/kg; 320mg/m² dose) and Cohort 3 (16 mg/kg; 640 mg/m² dose) is presented inTable 9. The mean AUC₀₋₂₄ is the relevant exposure for cetuximab-IR700,as the pharmacological activity is activated only through lightirradiation, which occurs at 24 hours post cetuximab-IR700 dosing.

TABLE 9 Pharmacokinetic data for dose escalation of Cetuximab-IRDye700DX conjugate Dosing Dosing Cetuximab-IR700 Mean (mg/kg) (mg/m²)AUC⁰⁻²⁴ (hr*μg/mL)  4.0 mg/kg 160 mg/m²  770 +/− 47.5  8.0 mg/kg 320mg/m² 1700 +/− 166  16.0 mg/kg 640 mg/m² 3,690 +/− 1,060

Table 9A summarizes the exposure of Erbitux® based on single doses asreported in Fracasso et al. (2007) Clin Cancer Res, 13(3), 986-993 (seeTable 3 therein). The FDA-approved dosing regimen for Erbitux® in thetreatment of head and neck cancer is an initial infusion of 400 mg/m²followed by weekly 250 mg/m² infusions. Based on the reported exposuresin Fracasso et al., the exposure for a 1-month treatment based on theFDA-approved dosing regimen for head and neck cancer was extrapolatedand is also listed in Table 9A.

TABLE 9A Exposure of Erbitux ® based on single doses and FDA-approveddosing regimen (see Fracasso et al. (2007) Clin Cancer Res, 13(3),986-993, Table 3) Dosing Erbitux Mean AUC_(0−inf)  50 mg/m² 858 +/− 271100 mg/m² 3,038 +/− 655   250 mg/m² 11,812 +/− 3,656  400 mg/m² 24,620+/− 9,555  500 mg/m² 24,740 +/− 8,259     400 mg/m² + 3 × 60,056(predicted) 250 mg/m² (1 month treatment)

As shown in Table 9A, the AUC_(0-∞) value for Erbitux® at the 400 mg/m²and the 250 mg/m² doses are reported to be approximately 24,620 μg/mL*hand 11,812 μg/mL*h, respectively, and are predicted to be approximately60,056 μg/mL*h following infusion according to the FDA-approved dosageregimen. In contrast, the exposure of cetuximab-IRDye 700DX followingadministration in accord with the above dosage regimen described in thisExample is much lower. The results show that the mean AUC₀₋₂₄, even atthe highest dose used in the current study (640 mg/m²), wasapproximately 15% of the AUC for 400 mg/m² Erbitux® (3,690 vs. 24,740μg/mL*h, respectively). Thus, these results demonstrate that the singledose of cetuximab-IRDye 700Dx conjugate resulted in exposure ofcetuximab that was far lower than the reported exposure observed to asingle dose at the therapeutic higher doses of Erbitux®.

Further, the single dose of cetuximab-IRDye 700DX conjugate administeredin this study was far lower than the dose of cetuximab that has beenapproved by the FDA for treatment of head and neck cancer. For example,the approved dose of Erbitux® to treat head and neck cancer is aninitial dose of 400 mg/m² followed by weekly administration of 250mg/m², and the mean AUC₀₋₂₄, even at the highest dose used in thecurrent study (640 mg/m²), was approximately 6% of the extrapolated AUCthe 1-month treatment with Erbitux® according to the FDA-approved dosageregimen (3,690 vs. 60,056 μg/mL*h, respectively).

The results showed that even at this reduced exposure, cetuximab-IRDye700DX conjugate elicited a rapid and effective tumor response upon lightactivation at 24 h post infusion, in all patients in the study. Thus,despite the lower exposure, the results demonstrated that a single dosetreatment with cetuximab-IRDye 700DX conjugate followed by irradiationresulted in a rapid and robust tumor response.

3. Trial of Photoimmunotherapy (PIT) with Repeated Cetuximab-IRDye 700DXAdministration

A treatment regimen is tested to determine the safety and efficacy ofrepeated treatment with administration of cetuximab-IRDye 700DXconjugate followed by photoimmunotherapy (PIT). Up to twenty (20) adultmale and female patients that have confirmed recurrent squamouscarcinoma of head and neck that cannot be satisfactorily treated withsurgery, radiation or platinum chemotherapy are included in the study.Patients included have received prior systemic platinum-basedchemotherapy unless contraindicated, have a life expectancy of greaterthan 6 months, and have an Eastern Cooperative Oncology Group (ECOG)performance score of 0 to 2.

Selected patients are administered with a single dose of 640 mg/m²cetuximab-IRDye 700DX conjugate via IV infusion over 2 hours on Day 1 ofthe beginning of treatment. To induce photoimmunotherapy (PIT), onelight application with a light having a wavelength of 690 nm wasperformed at 24 hours±3 hours (Day 2) post conjugate administration, ata fluence of 50 J/cm² for superficial illumination or 100 J/cm fiberlength for interstitial illumination. Follow-up observations are made at1 week, 2 weeks, and 1 month after the initial treatment (Treatment 1).For long-term follow-up, observations are made every 3 months for 2years following Treatment 1. Patients with remaining residual tumor four(4) weeks after Treatment 1 receive Treatment 2, with same dose and PITregimen. Patients with remaining residual tumor four (4) weeks afterTreatment 2 receive a further Treatment 3, maintaining the same dose andPIT regimen. Patients with remaining residual tumor four (4) weeks afterTreatment 3 receive a further Treatment 4, maintaining the same dose andPIT regimen, for up to a total of four (4) treatments.

Primary endpoint for the study is safety associated with up to four (4)repeated treatments. Secondary endpoints include evaluation ofpharmacokinetic parameters of the cetuximab-IRDye 700DX conjugate, tumorresponse evaluation using RECIST 1.1 Criteria at 1 month after lasttreatment via computerized tomography (CT) and/or positron emissiontomography (PET) scan, evaluation of long-term outcomes for overallsurvival (OS) and progression-free survival (PFS), and assessment ofdevelopment of anti-drug antibodies and neutralizing antibodies.

Example 3: Comparison of Conjugate Concentration to Alter Activity ofCell Surface Protein Versus to Mediate Cell Killing byPhotoimmunotherapy in In Vitro Assays

In vitro assays were employed to compare the effect of an IRDye700DX-antibody conjugate upon binding to its cell surface proteinantigen in functional assays for protein activity and inphotoimmunotherapy (PIT) assays. The assays were performed using theexemplary cetuximab-IRDye 700Dx conjugate, which specifically binds tothe cell surface protein epidermal growth factor receptor (EGFR) via theanti-EGFR antibody cetuximab. Similar in vitro assays are within thelevel of a skilled artisan to perform to assess other IRDye700DX-targeting molecule conjugates, e.g., IRDye 700DX-antibodyconjugates, using cell lines that express the protein to which thetargeting molecule (e.g., antibody) binds and/or assays that assess afunctional activity induced upon such binding.

A. In Vitro Phosphorylation Assay to Assess Functional Activity UponBinding EGFR

To assess the effects of cetuximab-IRDye 700Dx conjugate on regulatingfunctional activity of EGFR, a phosphorylation assay was performed. EGFis a natural ligand for EGFR that induces phosphorylation of EGFR uponbinding. A431 cells (ATCC CRL 1555) were plated in wells of a 96-wellplate and then were pre-incubated for 5 minutes with increasing amountsof cetuximab or cetuximab-IRDye 700DX conjugate. Cells were thenstimulated with 100 ng/mL EGF ligand for 10 min to inducephosphorylation of EGFR. The cells were fixed in the assay plate withformaldehyde and incubated with an anti-phospho-EGFR-specific antibody.After washing, phosphorylation of EGFR was measured by colorimetricquantification following addition of an anti-phospho-EFGR antibodyconjugated to HRP in an ELISA assay. Stop solution was added and theoptical density (OD) of the wells was measured in a microplate readerset to 450 nm. As a positive control, cells were stimulated with EGF inthe absence of any added cetuximab or cetuximab-IRDye 700DX conjugate(designated “NT”), and the phosphorylation (as indicated by the OD)induced in the positive control was set at 100%. The relative percent(%) phosphorylation of EGFR was determined as a percent of thephosphorylation of the positive control.

As shown in FIG. 1, cetuximab and cetuximab-IRDye 700DX conjugate bothprevented EGF-induced phosphorylation with equal potency. The inhibitoryeffect of cetuximab and cetuximab-IRDye 700DX was dose-dependent, withan IC₅₀ of 694 and 570 ng/mL, respectively. Thus, the results of theIn-Cell ELISA assay showed that, like cetuximab, the cetuximab-IRDye700DX conjugate also is able to bind to EGFR at or near the EGF bindingsite to prevent the stimulation and phosphorylation of EGFR by EGF.Since antibody binding is rapid and nearly irreversible, it is likelythat the potency curve for prevention of phosphorylation closelyreflects receptor occupancy by the antibody.

B. In Vitro Photoimmunotherapy (PIT) Assays

As a comparison to EGFR-induced phosphorylation, in vitro PITexperiments were performed for the cetuximab-IRDye 700DX conjugate. A431cells were incubated with increasing concentrations of cetuximab-IRDye700DX conjugate for 2 hours at 37° C. Then, 8 J/cm⁻² of 690 nm light wasapplied to induce PIT. To monitor the progression of cell death afterlight treatment, the conjugate containing medium was replaced by freshmedium containing CellTox Green (Promega), a dye that reports cell deathbased on compromised cell membrane integrity. Time dependent increase incell death was observed over 24 hours. As a control, cell death also wasassessed in cells in which cetuximab-IRDye 700DX conjugate was not added(designated “NT”).

As shown in FIG. 2, the extent of cell death was dose-dependent. Thehalf maximal cell death (EC₅₀) resulting from PIT with thecetuximab-IRDye 700DX conjugate was 37.6 ng/mL. Thus, this result showedthat the concentration of cetuximab-IRDye 700DX conjugate required toinduce half maximal cell death by PIT was more than 15 times lower thanthe concentration required to block half the phosphorylation in theELISA experiment described above (570 ng/mL). Additionally, thecetuximab-IRDye 700DX conjugate concentration that led to 90% cell death(257 ng/mL) resulted only in about 25% reduction of phosphorylation.

Similar experiments were performed with BxPC3 cells, in which PITinduced by increasing titrating doses of the cetuximab-IRDye 700DXconjugate was compared at two higher light doses (16 J/cm⁻² and 32J/cm²). As shown in FIG. 3, the results also showed the potency of thecetuximab-IRDye 700DX conjugate for inducing PIT even at very lowconcentrations. In particular, the results showed that the EC₅₀resulting from PIT with the cetuximab-IRDye 700DX conjugate was evenlower, which likely is due to the higher light doses that were applied.For example, increasing the light dose from 16 J/cm² to 32 J/cm²resulted in a decrease in the half maximal cell death (EC₅₀) from 22.6to 13.7 ng/mL. Thus, the results showed that higher light doses mayrequire even lower concentrations of cetuximab-IRDye 700DX conjugate(that means lower receptor occupancy) for the same extent of cellkilling.

Example 4: Synthesis and Evaluation of a Panitumunab-IRDye700DX-Alexa-488 Dual Label Conjugate

A dual-label conjugate was prepared in which the exemplary antibodypanitumunab was conjugated both to IRDye 700DX and Alexa Fluor 488.

A. Materials

The water-soluble, silicon-phthalocyanine derivative, IRDye 700DX NHSester (IR-700; C₇₄H₉₆N₁₁₂Na₄O₂₇S₆Si₃, molecular weight of 1954.22), wasobtained from LI-COR Bioscience (Lincoln, Nebr.). Alexa Fluor 488carboxylic acid, succinimidyl ester Alexa Fluor 488-NETS was obtainedfrom Life Technologies (Carlsbad, Calif.). Panitumumab, a fullyhumanized IgG2 mAb directed against the human epidermal growth factorreceptor 1 (HER1), was purchased from Amgen (Thousand Oaks, Calif.). Allother chemicals used were of reagent grade.

B. Synthesis of Panitumunab-IRDye 700DX-Alexa-488 (Pan-IRDye700DX-Alexa-488)

Panitumumab (2 mg, 13.6 nmol) in 0.1 mol/L Na₂HPO₄ (pH 8.5) wasincubated with 7 μL of a 5 mM DMSO solution of Alexa Fluor 488-NHS (35nmol) for 45 minutes in the dark at room temperature, whereupon IR-700DX(66.8 μg, 35.0 nmol, 5 mmol/L in DMSO) was added. After an additionalincubation time of 45 min at room temperature, 15 μL of 100 mM Tris basewas added to stop the conjugation reactions. Extraneous dye and othersmall molecule impurities were removed by filtration and excessivebuffer exchange (30 reaction volumes of HyCLone PBS pH=7.1 using Amicon®Ultra 15 Centrifugal Filter Units (Merck Millipore Ltd, Billerica,Mass.)).

C. Characterization of Conjugate

The conjugate was evaluated by size exclusion chromatography (SEC) on aTSKgel G2000 SWxl, 7.6×300 mm SEC column (TOSOH Biosciences, King ofPrussia, Pa.) using an Agilent 1100 HPLC system equipped with an AgilentG1315A diode array detector (DAD) monitoring the wavelengths of 280, 488and 690 nm, along with an Agilent G1321A fluorescence (FLS) detectormonitoring at the excitation wavelength at 488 nm and emissionwavelength at 505 nm.

The antibody concentration and average dye to antibody ratio (DAR) forboth dyes were determined using 280, 488 and 690 nm absorbance unitintegration area for the antibody monomer peak after the appropriateextinction coefficient value correction factors were applied as neededfor each respective dye. The DAR for the Alexa-488 and IRDye 700DX dyeswere calculated to be 1.0 and 2.1, respectively. The schematic structurefor the panitumumab-IRDye 700DXDX-Alexa-488 conjugate is shown in FIG.4.

The fluorescence results showed a strong Agilent HPLC signal, which wasobserved at 505 nm using an excitation wavelength of 488 nm for theAlexa Flour-488 conjugated antibody at the appropriate retention timefor an IgG1 150,000 dalton protein. In addition, a characteristicvisible green fluorescence was observed by eye from the Pan-IRDye700DX-Alexa-488 labeled conjugate using both ambient and 365 nmexcitation light (hand-held lamp).

D. Photoimmunotherapy (PIT) Using IRDye 700DX-Conjugated Antibodies

To evaluate whether photoimmunotherapy with IRDye 700DX-conjugatedantibody was impacted by the presence of an additional dye conjugated tothe same antibody, the PIT activity of the panitumunab-IRDye700DX-Alexa-488 dual label conjugate (Pan-IRDye 700DX-Alexa-488) wascompared to an anti-human EGFR antibody drug panitumumab conjugated onlywith IRDye 700DX (Pan-IRDye 700DX).

The two conjugates were tested for efficiency in killing cells in an invitro PIT assay using the pancreatic cancer line BxPC3. BxPC3 wereseeded into 96-well white-wall plates (4,000 cells/well) the day beforethe experiment. Antibody conjugates were added to cells at aconcentration of 10 μg/mL and were incubated for 2 hours at 37° C. Lightof 690 nm was applied by a diode at a constant power output of 50mW/cm². The illumination time was modulated to achieve the fluences 4J/cm², 8 J/cm² or 16 J/cm². As a control, a group of cells was notirradiated with light (0 J/cm²). To monitor the progression of celldeath after light treatment, the conjugate containing medium wasreplaced by fresh medium containing CellTox Green (Promega), a dye thatreports cell death based on compromised cell membrane integrity. Timedependent increase in cell death was observed over 24 hours.

As shown in FIG. 5A (Pan-IRDye 700DX) or FIG. 5B (Pan-IRDye700DX-Alexa-488), both conjugates led to near complete cell killing with16 J/cm² of light at 690 nm. Moreover, the sensitivity of the twoconjugates to light to induce killing was almost identical. A light doseof 8 J/cm² at 690 nm resulted in 74% and 73% killing by single(Pan-IRDye 700DX) and dual-labeled (Pan-IRDye 700DX-Alexa-488)panitumumab, respectively, and a light dose of 4 J/cm² at 690 nm wasonly marginally effective for both conjugates. Thus, the results showedthat the presence of the dye Alexa488 did not interfere with theabilities of a panitumumab-IRDye 700DX conjugate to induce 690 nmlight-dependent cell killing.

Example 5: Assessment of Cell Killing Activity and Composition ofVarious Antibody:IR700 DX Conjugates

Studies were performed to assess whether antibody-IRDye 700DX conjugatespre-exposed to different wavelengths of light differentially affectsoluble aggregate formation. Two different antibodies-mouse anti-humananti-PD-L1 (Catalog No: 329728, Biolegend, San Diego, Calif.) andanti-EGFR (cetuximab; Myoderm USA, Norristown, Pa.)—were labeled withIRDye 700DX and were evaluated to assess if pre-exposure to differentwavelengths of light affected soluble aggregate formation.

A. Antibody Conjugation

Both antibodies were conjugated with IRDye 700DX using the sameapproach. For all conjugates described below, the general protocol usedto conjugate the antibodies was similar to that of larger scaleconjugation with cetuximab-IRDye 700DX described in Example 1.Modifications to the protocol were made for smaller scale reactionvolumes that used 3 mg or less of protein.

The antibody solution (either anti-PD-L1 antibody or anti-EGFR antibody)was first exchanged with phosphate buffer saline pH 7 using a 30,000Dalton molecular weight cutoff centrifugal filter, then the antibodysolution pH was adjusted to a pH of 8.5 with addition of phosphatebuffer at pH=9. Frozen solid aliquots of IRDye 700DX NHS Ester (CatalogNo. 929-70011; Li-COR, Lincoln, Nebr.) were thawed at room temperature,then dissolved with DMSO to achieve a 10 mg/mL concentration. In a darkenvironment, the solubilized IR700 NHS Ester was then added to theantibody solution at a 4 (IR700 NHS Ester) to 1 (antibody) molar ratio.The conjugation reaction proceeded at 25° C. for 2 hours protected fromlight. Glycine (pH 8.2) was added to a final concentration of 10 mM for15 minutes to quench the reaction. The antibody conjugate solution wasthen exchanged with a 30,000 Dalton molecular weight cutoff centrifugalfilter with 24 mL of PBS pH 7 to remove free dye, glycine, andglycine-IR700, and to adjust the pH of the solution back to pH 7. Theantibody conjugates were analyzed with size exclusion chromatography toevaluate antibody-IR700 concentration, monomer purity, % solubleaggregate, and dye to antibody ratio (DAR).

B. Effects of Light Pre-Exposure on Composition of IRDye 700DX Conjugate

The antibody-IRDye 700DX conjugate was tested for formation of solubleaggregates under four different conditions with at least 30 μL ofconjugate at an antibody conjugate concentration of 850 μg/mL. The fourtreatment conditions were as follows: (1) antibody-IRDye 700DX conjugatestored at 4° C. protected from light (“4° C.” control); (2)antibody-IRDye 700DX conjugate placed in a clear glass HPLC tube under ahalogen lamp (Catalog No: PL-800, Dolan-Jenner, Boxborough, Mass.) at2500 lux for 24 hrs (“white light”); (3) antibody-IRDye 700DX conjugateplaced in a clear glass HPLC tube wrapped in aluminum foil to protectfrom light exposure under halogen lamp at 2500 lux for 24 hrs (“nolight”, used to control for thermal heating effects on the formation ofaggregates); and (4) antibody-IRDye 700DX conjugate placed in a clearglass HPLC tube and exposed to green LED lamp (Catalog No: Green-ECSGP19 EcoSmart) at 2500 lux for 24 hrs (“green light”). After 24 hoursunder each treatment condition, monomer purity and soluble aggregateformation was assessed by size exclusion chromatography.

The results for the anti-PD-L1-IRDye 700DX conjugate are shown in Table10. As shown, anti-PD-L1-IRDye 700DX conjugate (DAR ˜3) that was storedat 4° C. exhibited low soluble aggregate formation (<1.5%) and highmonomer purity (>96%) as measured by 280 nm absorbance and 690 nmabsorbance. Exposure of the anti-PD-L1-IRDye 700DX conjugate to 2500 luxof white light from a halogen lamp resulted in a significant increase insoluble aggregate formation (˜30%) and concomitant decrease in monomerpurity (˜65%) as measured by 280 nm absorbance and 690 nm absorbance.Anti-PD-L1 IRDye 700DX exposed to the thermal heating effects of 2500lux white light from a halogen lamp, but protected from lightillumination using aluminum foil, did not induce any increase in solubleaggregate formation when compared to that of the 4° C. control sample.Anti-PD-L1 IRDye 700DX conjugate exposed to light from a green LED lampresulted in a very minor increase in soluble aggregate formation (˜5%),which was a significantly lower amount of soluble aggregate formationthan that of anti-PD-L1 IRDye 700DX exposed to white light.

TABLE 10 Anti-PD-L1 IRDye 700DX aggregate formation with different typesof light exposures. Aggregate Monomer Retention Retention % Aggregate %monomer Treatment time [min] time [min] (Aggregate/Total)(Monomer/Total) 1) Anti-PD-L1-IRDye 7.2 8.3  1.2% (280 nm) 96.7% (280nm) 700DX: 4° C.  1.1% (690 nm) 98.4% (690 nm) 2) Anti-PD-L1-IRDye 6.87.6 30.9% (280 nm) 65.0% (280 nm) 700DX: 2500 Lux 29.5% (690 nm) 64.7%(690 nm) white light, 24 hours 3) Anti-PD-L1-IRDye 7.2 8.3  1.1% (280nm) 98.9% (280 nm) 700DX: No light, 24 hours  1.1% (690 nm) 98.2% (690nm) 4) Anti-PD-L1-IRDye 7.2 8.3  5.4% (280 nm) 94.6% (280 nm) 700DX:2500 Lux  5.1% (690 nm) 94.4% (690 nm) green light, 24 hours

The results for the cetuximab-IRDye 700DX conjugate are shown in Table11. Cetuximab-IRDye 700DX conjugates (DAR ˜3) that were stored at 4° C.did not have any detectable soluble aggregate formation (˜0%) and highmonomer purity (˜100%) as measured at 280 nm absorbance and 690 nmabsorbance. Exposure of the cetuximab-IRDye 700DX conjugate to whitelight of 2500 lux from a halogen lamp resulted in a significant increasein soluble aggregate formation (˜40%) and concomitant decrease inmonomer purity (˜55%) as measured by 280 nm absorbance and 690 nmabsorbance. Cetuximab-IRDye 700DX exposed to the thermal heating effectsof 2500 lux white light from a halogen lamp, but protected from lightillumination using aluminum foil, did not induce any increase in solubleaggregate formation when compared to that of the 4° C. control sample.Cetuximab-IRDye 700DX conjugate exposure to light from a green LED lampresulted in a minor increase in soluble aggregate formation (˜4%), whichwas significantly lower amount of soluble aggregate formation than thatof cetuximab-IRDye 700DX exposed to white light.

TABLE 11 Cetuximab-IRDye 700DX aggregate formation with different typesof light exposures. Aggregate Monomer Retention Retention % Aggregate %monomer Sample time [min] time [min] (Aggregate/Total)(Monomer/Total) 1) cetuximab-IRDye ND 8.2   0% (280 nm)  100% (280 nm)700DX: 4° C  0.2% (690 nm) 99.3% (690 nm) 2) cetuximab-IRDye 40.5% (280nm) 55.3% (280 nm) 700DX: 2500 Lux 7.1 7.9 41.8% (690 nm) 53.8% (690 nm)white light, 24 hours 3) cetuximab-IRDye  0.3% (280 nm) 99.6% (280 nm)700DX: No light, 24 hours 7.3 8.2  0.2% (690 nm) 99.6% (690 nm) 4)cetuximab-IRDye  3.9% (280 nm) 96.1% (280 nm) 700DX: 2500 Lux 7.3 8.2 3.5% (690 nm) 96.0% (690 nm) green light, 24 hours

Example 6: Duration of Pre-Exposure of White Fluorescent Vs. Green LEDLighting and their Effect on Cetuximab-IRDye 700DX Soluble AggregateFormation and PIT Potency

The following studies were performed to assess whether cetuximab-IRDye700DX conjugates pre-exposed to different wavelengths of light and fordifferent durations of exposure differentially affect soluble aggregateformation and pharmacological activity.

Cetuximab-IRDye 700DX was conjugated as described in Example 1. Thefollowing 14 different conditions were assessed: sample was exposed to500 lux white fluorescent lighting at 25° C. for different durations oflight exposure at 24 hours, 12 hours, 6 hours, 3 hours, 1.5 hours, and45 minutes; sample was exposed to 500 lux of green LED lighting (CatalogNo: Green-ECS GP19 EcoSmart) at 25° C. for different durations of lightexposure at 24 hours, 12 hours, 6 hours, 3 hours, 1.5 hours, and 45minutes; sample was exposed to no light at 25° C.; and sample wasexposed to no light at 4° C. The duration of the light exposure for 24hours, 12 hours, 6 hours, 3 hours, 1.5 hours, 45 minutes corresponds to12,000 lux-hours, 6,000 lux-hours, 3,000 lux-hours, 1,500 lux-hours, 750lux-hours, or 375 lux-hours, respectively. For each condition, 30 μL ofconjugate was placed in a clear HPLC vial per sample at an antibodyconjugate concentration of 2 mg/mL and the sample was exposed to eachlight condition.

The composition of cetuximab-IRDye 700DX conjugate following white lightor green light exposure for different durations of time was assessed bymonitoring formation of soluble aggregates and PIT killing activity.

1. Aggregate Formation

Cetuximab-IRDye 700DX was analyzed with HPLC size exclusionchromatography to evaluate the monomer purity and soluble aggregateformation. The percent soluble aggregate formation was measured as afunction of cetuximab-IRDye 700DX lux-hours exposure to white light,green light, or no light.

As shown in FIG. 6A, the duration of exposure of cetuximab-IRDye 700DXto 500 Lux white fluorescent light had a direct effect on the formationof soluble aggregates. Cetuximab-IRDye 700DX exposure to whitefluorescent light resulted in a rapid increase in soluble aggregateformation with the presence of greater than 5.0% soluble aggregateformation observed even after only 375 lux-hours (45 minutes at 500 lux)of exposure to white light, which increased further with the increasedduration of exposure to white fluorescent lighting. Cetuximab-IRDye700DX green light exposure also slightly increased soluble aggregateformation albeit at a rate much slower than that of white light; thepercentage of aggregates formed even after exposure to 12,000 lux-hours(24 hours at 500 lux) of green light was no more than 5.0%. The resultsshowed that there was a greater cetuximab-IRDye 700DX soluble aggregateformation with an increase in time of exposure to white light than thatof green light. Less than 1% soluble aggregate formation was observed insamples either incubated at 4° C. or 25° C. when protected from anylight exposure.

2. PIT Killing

To evaluate PIT killing activity by the cetuximab-IRDye 700DXpre-exposed to the various light conditions, BxPC3 cells (#CRL-1687,ATCC, Manassas Va.) were incubated for one hour at 4° C. with or without1 μg/mL cetuximab-IRDye 700DX in RPMI-1640 media supplemented with 10%FBS and 1% Penicillin/Streptomycin (complete culture media), and thenwashed one time with complete culture media to remove unboundcetuximab-IRDye 700DX. The cells were then illuminated with a 690 nmlaser at a light dose of 32 J/cm² or protected from light (0 J/cm²).

The effect of different treatment regimens on cell death was measuredusing the fluorescent stain, CellTox Green (Cat No: G8731, Promega,Madison, Wis.). CellTox Green is a non-permeable fluorescent dye thatexhibits increased fluorescence upon binding to DNA. Therefore, onlycells that have compromised plasma membranes exhibit strong CellToxGreen staining. After the light treatment, all cells were incubated with1× CellTox Green reagent diluted in RPMI-1640 supplemented with 10%fetal bovine serum and 1% Penicillin/Streptomycin (complete culturemedia). Wells that did not include any cells were also incubated with 1×CellTox Green reagent diluted in complete culture media to serve asbackground subtraction wells during fluorescent signal detection. TheCellTox Green fluorescence signal was measured at 24 hours after lighttreatment using a fluorescence plate reader. The cells were then lysedwith detergent, incubated at 37° C. for 30 minutes, and the CellToxGreen fluorescence signal was measured again post lysis. The percentdead cells was calculated by taking the ratio between background (1×CellTox Green in complete culture media without cells) subtractedCellTox Green signal per well prior to and post lysis and multiplyingthe ratio by 100.

As shown in FIG. 6B, no effect on cell death was observed for allsamples exposed to 0 J/cm⁻² during the PIT treatment, indicating that,despite the increase in soluble aggregates after pre-exposure to whitelight, the soluble aggregates were not cytotoxic in that absence oflight irradiation. In contrast, cell killing was observed for samplesthat were subsequently irradiated with a 690 nm laser at a light dose 32J/cm⁻², although the extent of cell killing was substantially reduced bythe cetuximab-IRDye 700DX exposed to increased durations of white light.As shown, cetuximab-IRDye 700DX pre-exposed to 3,000 Lux-Hours (500 luxfor 6 hours) or more of white fluorescent light exhibited less than 90%or less effect on PIT activity. However, cetuximab-IRDye 700DX exposedto all lux-hour doses of green light evaluated did not result in aneffect in PIT potency, indicating that pre-exposure to green light didnot substantially impact light-activated killing activity.

The effect of aggregate formation on PIT activity is shown in FIG. 6C.As shown, the PIT potency (percent dead cells) for all cetuximab-IR700treatment regimens for evaluating white light and green light exposurewere plotted as a function of the measured percent soluble aggregate foreach respective sample. The results showed that greater than 15% solubleaggregate formation of cetuximab-IRDye 700DX results in a significantdecrease in PIT potency.

Example 7: Effect of Indirect Conjugation with Phthalocyanine Dye on PITKilling and Specificity of PIT

The following studies were performed to assess whether antibodies thatbind directly to cell surface molecules require direct conjugation witha phthalocyanine photosensitizer, such as IRDye 700DX, to mediate PITkilling activity.

A. IR700 Conjugation of Secondary Antibody Against Cell TargetingAntibody

Instead of directly conjugating a targeting antibody targeted against acell surface molecule (e.g. on a cancer cell) with IR700, a secondaryanti-human IgG antibody that bound the targeting antibody was conjugatedwith IR700. Specifically, AffiniPure Donkey Anti-Human IgG, Fcγ FragmentSpecific (DxHu) antibody (Catalog number: 709-005-098, JacksonImmunoResearch Laboratories, West Grove, Pa.) was labeled with IRDye700DX to evaluate whether non-covalent labeling of primary antibodieswith secondary antibody-IRDye 700DX could be used in PIT-mediatedkilling. The protocol used for conjugating the DxHu antibody with IRDye700DX was substantially the same as the protocol for antibodyconjugation used in Example 5.

PIT killing of BxPC3 cells was evaluated similar to the method describedin Example 6, except the cells were first incubated for one hour at 4°C. with or without anti-EGFR antibody, cetuximab (Myoderm USA,Norristown, Pa.) in RPMI-1640 media supplemented with 10% FBS and 1%Penicillin/Streptomycin (complete culture media). The cells were thenwashed one time with complete culture media, incubated for 30 minutes at4° C. with or without IRDye 700DX conjugated (DxHu IRDye 700DX)secondary antibody diluted with complete culture media, and then washedone time with complete culture media. As a control, BxPC3 cells wereincubated with cetuximab-IRDye 700DX in which the cetuximab was directlyconjugated to IRDye 700DX. To induce cell killing, the cells were thenilluminated with a 690 nm laser at a light dose of 16 J/cm⁻² orprotected from light (“no light”). Cell death was evaluated as describedin Example 6 using CellTox Green.

As shown in FIG. 7, BxPC3 cells that were sequentially labeled withcetuximab and donkey anti-human IRDye 700DX secondary antibody andtreated with light exhibited ˜90% cell death. The same treatment withthe primary and secondary antibody did not result in cell death whencells were not exposed to the 690 nm light treatment. Light illuminationof cells treated only with the secondary antibody did not lead to celldeath because the DxHu IRDye 700DX secondary antibody does not binddirectly to cells in the absence of pre-incubation with a human-derivedprimary antibody targeting a cell surface antigen. The extent of cellkilling induced by sequential exposure to the antibodies was evenslightly greater than in BxPC3 cells incubated with cetuximab that hadbeen directly labeled with IRDye 700DX. Light treatment of BxPC3 cellstreated only with media alone with no incubation with either cetuximabor DxHu IRDye 700DX resulted in a basal cell death level of ˜10%, whichwas similar to the background cell death in cells that were notirradiated with light (no light treatment). Thus, the results showedthat antibodies that bind directly to cancer cells do not require directconjugation of a phthalocyanine photosensitizer such as IRDye 700DX tomediate PIT killing activity. Indirect labeling of anti-cancerantibodies mediated by a secondary antibody conjugated IRDye 700DX canalso induce effective PIT killing activity.

B. IR700 Conjugation of Monomeric Streptavidin Against a BiotinylatedCell Targeting Antibody

In another study, the PIT killing activity of cells sequentiallyincubated with a biotinylated anti-EGFR antibody (biotinylatedcetuximab) and monomeric streptavidin-conjugated IRDye 700DX wasexamined. Furthermore, the effect of pre-exposure of the monomericstreptavidin-IR700 to white light on the PIT killing activity was alsoexamined.

1. Conjugations

a. Conjugation of Biotin to Cetuximab

To conjugate the anti-EGFR antibody cetuximab to biotin, a 5 mL volumeof anti-EGFR antibody (cetuximab; Myoderm USA, Norristown, Pa.) suppliedat a concentration of 2 mg/mL in PBS pH 7.2 was concentrated to a volumeof 2 mL (5 mg/mL) using a 30,000 Dalton molecular weight cutoffcentrifugal filter (Cat No: UFC903024, Merck-Millipore, Cork, IRL.) Thesolution was diluted to 5 mL with 100 mM Na₂HPO₄ (pH 8.9) to finalvolume of 5 mL and pH of ˜8.5.

EZ-Link Sulfo-NHS-LC-Biotin (sulfosuccinimidyl-6[biotin-amido]hexanoate)was used to label the antibody according to the manufacturer'sinstructions (Cat. No. 21327, ThermoScientific, Rockford, Ill.).Specifically, a 2 mg sample of Sulfo-NHS-Biotin (503-biotin-NHS ester,Cat #: 1854200, Thermo Scientific) was thawed at room temperature, thendissolved with deionized (DI) water to achieve a 10 mg/mL concentration.A volume of 130 μL of the solubilized 503-biotin-NHS ester was added tothe cetuximab antibody solution at a 20 (SO₃-Biotin-NHS Ester) to 1(cetuximab antibody) molar ratio. The conjugation reaction proceeded at25° C. for 2 hours protected from light where upon, excess glycine wasadded to quench the reaction for 15 minutes. The cetuximab-biotinconjugate solution was then exchanged with ten times the equivalentconjugation volume with PBS pH 7.2 using a 30,000 Dalton molecularweight cutoff centrifugal filter to remove free dye, glycine, andglycine-IRDye 700DX, and to adjust the pH back to pH 7.2.

The cetuximab-biotin conjugate was analyzed with size exclusionchromatography (SE-HPLC) to evaluate monomeric cetuximab-biotin purity,% soluble aggregate and reaction product residual impurity levels. Theaverage molar Biotin to Antibody Ratio (BAR) for the conjugate wasdetermined using the Pierce Colorimetric Biotin Quantification Assay(Cat No: 128005, Thermo Scientific, Rockford, Ill.) according tosupplier instructions. The results are shown in Table 12.

TABLE 12 Cetuximab-Biotin Analysis Results Biotin to Antibody Ratio 7.2(BAR) SE-HPLC A210 99.1% monomer, 0.3% HMW, 0.6 LMW Purity A280 100 %

b. Conjugation of Monomeric Streptavidin to IR700

The general protocol used to conjugate engineered monomeric streptavidin2 (mSA2) (Catalog No: EBU001/2, Kerafast, Boston, Mass.) with IR700 wassubstantially the same as the protocol for antibody conjugationdescribed in Example 5, except that prior to conjugation, the mSA2solution was first exchanged with phosphate buffer saline pH 7 using a3,000 Dalton molecular weight cutoff centrifugal filter. For theconjugation, the solubilized IR700 NHS Ester was then added to the mSA2solution at a 2 (IR700 NHS Ester) to 1 (monomeric streptavidin) molarratio. After the conjugation reaction performed substantially asdescribed in Example 5, the monomeric streptavidin conjugate solutionwas then exchanged with 24 mL of PBS pH 7 using a 10,000 Daltonmolecular weight cutoff centrifugal filter to remove free dye, glycine,and glycine-IR700, and to adjust the pH back to pH 7.

2. PIT Killing

Biotinylated cetuximab was pre-incubated with monomericstreptavidin-IRDye 700DX at a 20 (monomeric streptavidin IRDye 700DX) to1 (1 μg/mL biotinylated cetuximab) molar ratio for 1 hour at roomtemperature. BxPC3 cells were incubated with RPMI media supplementedwith 10% FBS and 1% Penicillin/Streptomycin (complete culture media)containing 1 μg/mL of biotinylated cetuximab pre-complexed withmonomeric streptavidin-IRDye 700DX or complete culture media only forone hour at 37° C. The cells were then washed one time with completeculture media. The cells were either protected from light (light dose 0J/cm²) or were illuminated with a 690 nm laser with different lightdosimetries (2 J/cm², 8 J/cm², 32 J/cm² or 64 J/cm²). Cell death wasevaluated as described in Example 6 using CellTox Green.

As shown in FIG. 8A, the light-dependent PIT killing activity of BxPC3cells with biotinylated cetuximab pre-complexed with monomericstreptavidin-IRDye 700DX (mSA IRDye 700DX) was light dose dependent. Nolight-dependent killing activity was observed with cells incubated withcomplete culture media alone.

To confirm specificity of the effect, the effect of biotinylatedcetuximab pre-complexed with monomeric streptavidin-IRDye 700DX wasevaluated in the presence of either unconjugated cetuximab orunconjugated monomeric streptavidin to assess if the effect could becompeted. In one condition, BxPC3 cells were first pre-incubated with100 μg/mL unconjugated cetuximab or complete culture media alone for onehour at 37° C. The cells were then washed one time. The cellspre-incubated with unconjugated cetuximab were then incubated withcomplete culture media containing 1 μg/mL biotinylated cetuximabpre-complexed with 2 μg/mL monomeric streptavidin IRDye 700DX. Inanother condition, cells that had been pre-incubated with completeculture media alone (but not preincubated with unconjugated cetuximab)were incubated with 1 μg/mL biotinylated cetuximab that had beenpre-complexed in the presence of 10-fold excess unconjugated monomericstreptavidin (complexing performed with 20 μg/mL unconjugated monmericstreptavidin and 2 μg/mL monomeric streptavidin IRDye 700DX). Inaddition, cells that had been preincubated with cell culture media (butnot preincubated with unconjugated cetuximab) either incubated with 2μg/mL monomeric streptavidin IRDye 700DX alone or complete culture mediaonly for one hour at 37° C. The cells were then washed one time withcomplete culture media.

The results shown in FIG. 8B demonstrated the PIT-mediated killing withbiotinylated cetuximab pre-complexed with monomeric streptavidin-IRDye700DX (mSA IRDye 700DX) was specific to cells having bound cetuximabassociated with IR700. No light-dependent PIT killing was observed whenBxPC3 cells were pre-exposed to 100 μg/mL unconjugated cetuximab priorto incubation with biotinylated cetuximab pre-complexed with monomericstreptavidin-IRDye 700DX. The results also showed that the PIT killingwas dependent on the association of the IR700 conjugated monomericstreptavidin and biotinylated antibody, since no light-dependent PITkilling of BxPC3 cells incubated with biotinylated cetuximabpre-complexed with 10x molar excess of unconjugated monomericstreptavidin over monomeric streptavidin-IRDye 700DX was observed.Further, the results demonstrated that no light-dependent PIT killing ofBxPC3 cells was observed in cells incubated with monomericstreptavidin-IRDye 700DX alone in the absence of biotinylated cetuximabor BxPC3 cells incubated in culture media alone.

3. Effects of Light Pre-Exposure on Composition and Activity

The effect of indirect killing of cells using monomericstreptavidin-IRDye 700DX that had been exposed to different types oflight was also evaluated. Thirty microliters of monomericstreptavidin-IRDye 700DX conjugate (DAR 1.35) was added per clear HPLCvial at a monomeric streptavidin conjugate concentration of 865 μg/mL.The following conditions were tested: (1) the monomericstreptavidin-IRDye 700DX conjugate was placed in a clear glass HPLC tubewrapped in aluminum foil to protect from light exposure under a halogenlamp at 2500 lux for 24 hrs (“no light”; to control for thermal heatingeffects); (2) the monomeric streptavidin-IRDye 700DX conjugate wasplaced in a clear glass HPLC tube under a halogen lamp at 2500 lux for24 hrs (“white light”); (3) the monomeric streptavidin-IRDye 700DXconjugate was placed in a clear glass HPLC tube and exposed to green LEDlamp at 2500 lux for 24 hrs (“green light”).

Cell killing induced by the monomeric streptavidin-IRDye 700DXpre-exposed under the various conditions and that had been complexedwith biotinylated cetuximab was assessed on BxPC3 cells as describedabove. Thus, all BxPC3 cell treatments were incubated with eithercomplete culture media or complete culture media containing biotinylatedcetuximab pre-complexed monomeric streptavidin-IRDye 700DX that hadundergone pre-exposure to light of different wavelengths of light asdescribed above.

As shown in FIG. 8C, the results revealed that monomericstreptavidin-IRDye 700DX pre-exposure to white light inhibits potentialfor PIT killing activity. The expected light-dependent killing of BxPC3cells was observed when cells were incubated with biotinylated cetuximabpre-complexed with monomeric streptavidin-IRDye 700DX that had beenprotected from light exposure with aluminum foil. In contrast, nolight-dependent PIT killing of BxPC3 cells was observed when cells wereincubated with biotinylated cetuximab pre-complexed with monomericstreptavidin that had been exposed to white light from a halogen lamp at2500 lux for 24 hours.

The results showed that the loss of PIT killing upon light exposure wasreduced when BxPC3 cells were incubated with biotinylated cetuximabpre-complexed with monomeric streptavidin-IRDye 700DX that had beenexposed to green light from a green LED lamp at 2500 lux for 24 hours,although in this experiment there was some decrease in PIT killing evenwhen the IR700 conjugate was pre-exposed to green light. Nolight-dependent PIT killing of BxPC3 cells incubated with completeculture media alone.

Example 8: Effect of Anti-EpCAM Antibody-IR700 Conjugate on PIT Killing

A further additional study was performed to assess the effect on cellkilling of an anti-mouse CD326 (EpCAM) (Catalog No: 118202, BioLegend,San Diego, Calif.) conjugated to a phthalocyanine photosensitizer suchas IRDye 700DX. The antibody targets a further alternative cell surfacemolecule, EpCAM. To prepare the anti-EpCAM-IRDye 700DX, conjugation wasperformed as described in Example 5.

To evaluate PIT killing activity by the anti-EpCAM-IRDye 700DXconjugate, 4T1 cells were incubated with RPMI media supplemented with10% FBS and 1% Penicillin/Streptomycin (complete culture media)containing increasing concentrations of anti-EpCAM-IRDye 700DX asindicated or complete culture media only for one hour at 37° C. Thecells were then washed one time with complete culture media. The cellswere then illuminated with a 690 nm laser at 0 or 32 J/cm² lightdosimetries. Cell death was evaluated as described in Example 6 usingCellTox Green.

As shown in FIG. 9A, the results showed that 4T1 cells incubated withanti-EpCAM-IRDye 700DX and illuminated at 32 J/cm² were killed in anantibody dose dependent manner. No significant cell death was observedat any antibody concentration without light illumination.

To confirm specificity of the cell killing, 4T1 cells were incubatedwith a molar excess unconjugated anti-EpCAM antibody to block binding ofthe anti-EpCAM-IRDye 700DX conjugate to the cell surface. Specifically,10, 1, or 0.1 μg/mL unconjugated anti-EpCAM antibody or complete culturemedia alone for one hour at 37° C. Without washing the cells,anti-EpCAM-IRDye 700DX was added to 4T1 cells to achieve a finalconcentration of 0.1 μg/mL and incubated for one hour at 37° C. Cellkilling was induced by illumination with a 690 nm laser at a 32 J/cm²light dose and cell killing determined using CellTox Green as describedabove.

The results are shown in FIG. 9B, which shows the specificity ofanti-EpCAM-IRDye 700DX PIT killing activity. The results showed that 4T1cells that were pre-incubated with unconjugated anti-EpCAM antibodyprior to incubation with anti-EpCAM-IRDye 700DX displayed significantlyless cell death after exposure to 32 J/cm² laser illumination incomparison to the 4T1 cells that did not undergo the blocking step,demonstrating that cell binding of anti-EpCAM and conjugation with IRDye700DX is necessary for photoimmunotherapy-based killing.

Example 9: PIT Killing of Fc Receptor-Expressing Target Cells withCetuximab-IRDye 700DX

The following studies were performed to assess whether antibody-IRDye700DX drug conjugate can bind to Fc receptor (FcR) and whetheractivation with near infrared (˜690 nm) light results in FcR+ cellkilling. FcR are commonly found on wide variety of immune cells such as,monocytes, macrophages and myeloid derived suppressor cells (MDSCs). Therole of these cells in solid tumors have been found to be detrimentaland tumor promoting. Human monocytic cell line THP1 express surface Fcreceptors and was used as the model cell system for this assay.

THP1 cells (ATCC, TIB-202) grown in complete RPMI 1640 medium wereplated at 5000 cells in 100 μL total volume per well in a 96 well tissueculture plate for adherence overnight. The viability of the cells priorto plating was checked via trypan blue exclusion method and >95% cellswere viable. The cells were divided into three groups (all intriplicate) as follows: (1) THP1 cells only (untreated); (2) THP1 cellstreated with the drug cetuximab-IRDye 700DX at 500 ng/mL; and (3) THP1cells first incubated with Fc receptor blocking solution (Catalog No:564220, BD, Franklin Lakes, N.J.) at 1 μg/well for 20 min at roomtemperature followed by treatment with drug cetuximab-IRDye 700DX (500ng/mL, 1 hr at 37° C. in incubator protected from light).

To induce killing, cells in each group were subjected to 690 nm laserlight at a dose of 32 J/cm⁻². The controls represented wellscorresponding to the groups described above but not treated with light.Cell killing was assessed using CellTox Green as described substantiallyas described Example 6. CellTox Green dye (1×) was added to the wellsand cells were incubated for 24 hours at 37° C. in an incubator. The dyewas also added to couple of wells just containing 100 μL of the mediumfor background subtraction later. After the incubation, the tissueculture plate was immediately read on a plate reader. The cells werethen subjected to lysis by adding 5 of diluted lysis solution (Promega,cat #G1821) including also the control wells containing just the media.The dilution was performed by adding culture medium to the lysissolution at 40% (lysis solution): 60% (culture medium) ratio. The platewas then read again to obtain values for 100% cell death. For each read,the two background wells were averaged and their values subtracted fromall other wells. In order to calculate the % cell death for each well,the background subtracted value from the first read was divided by thevalue from the second read (after lysis), and multiplied by 100.

As shown in FIG. 10, the results showed the Fc receptor-specific killingof THP1 cells by cetuximab-IRDye 700DX. Maximum killing was observed inthe group represented by drug treated THP1 cells subjected to 32 J/cm⁻²light. The percent killing values are relative to the light and drugtreated THP1 cells. Thus, the results showed that antibody-mediatedkilling can be mediated by specific binding to target molecules on thecell surface as well as, in some cases, binding of the antibody to theFcR.

Example 10: Assessment of Cell Killing Activity and Effect of WhiteLight Exposure on Cell Killing Activity of Non-Antibody Molecule:IR700DX Conjugates

The following studies were performed to assess if non-antibody proteins,small proteins, and viruses can be conjugated with a phthalocyanine dye,such as IR700, to target cell killing. As shown below, the resultsshowed that various other non-antibody molecules mediate cell killingthat is dependent on activation with near infrared light (e.g. about 690nm light), binding to cells, and/or affected by pre-exposure of thetargeting molecule conjugate to white light.

A. Non-Antibody Protein:IR700 Conjugate

Human recombinant epidermal growth factor (EGF) (Catalog No: 01-401, EMDMillipore, Billerica, Mass.) was conjugated to IRDye 700DX and evaluatedto assess its killing activity and if pre-exposure to differentwavelengths of light affected soluble aggregate formation.

1. EGF Conjugation

The protocol used for labeling of the human recombinant EGF with IRDye700DX was substantially the same as the protocol for antibodyconjugation described in Example 5, except that the prior toconjugation, the EGF solution was first exchanged with phosphate buffersaline pH 7 using a 3,000 Dalton molecular weight cutoff centrifugalfilter. For the conjugation, the solubilized IR700 NHS Ester was thenadded to the EGF solution at a 4 (IR700 NHS Ester) to 1 (EGF) molarratio or at a molar ratio of 1.2 (IR700 NHS Ester) to 1 (EGF). After theconjugation reaction performed as described in part A, the EGF conjugatesolution was then exchanged with six times the equivalent conjugationvolume with PBS pH 7 using a 3,000 Dalton molecular weight cutoffcentrifugal filter to remove free dye, glycine, and glycine-IR700, andto adjust the pH back to pH 7.

2. EGF-IR700 Light-Dependent Killing Activity

Photo-activated EGF-IR700 cell killing was assessed in A431 cells. A431cells were seeded at 5000 cells per well in 96 well white clear bottomdishes one day prior to the experiment. The following day, the A431cells were washed three times with EMEM supplemented with 1%Penicillin/Streptomycin (serum free media). The A431 cells were thenwashed one time with serum free media, then incubated with serum freemedia containing 1 μg/mL of EGF-IRDye 700DX for one hour at 4° C. orserum free media only. As a control to assess the specificity of theactivity, in one condition A431 cells were pre-incubated with 100 μg/mLunconjugated cetuximab diluted in serum free media for one hour at 4° C.prior to incubation with 1 μg/mL of EGF-IRDye 700DX. Cetuximab is acompetitive inhibitor of EGF binding to EGFR. The cells were then washedone time with serum free media.

To induce IR700-dependent killing, the cells were then illuminated witha 690 nm laser with 32 J/cm² of light or protected from light (“nolight”). Cell death was evaluated as described in Example 6 usingCellTox Green. The normalized percentage of dead cells was calculated bysubtracting all wells by the percentage of dead cells from the no lightserum free media only control, dividing by EGF-IRDye 700DX at 32 J/cm²minus the no light serum free media only control, and multiplied 100.

As shown in FIG. 11A, the results showed that EGF-IRDye 700DX mediatedcell killing is light-dependent killing with killing observed only whencells were treated with light to activate cell killing activity.Pre-exposure of A431 cells with 100 μg/mL unconjugated cetuximab priorto incubation with 1 μg/mL EGF-IRDye 700DX blocked light-dependent cellkilling. A431 cells incubated with media alone did not exhibit anylight-induced killing.

3. Effects of Light Pre-Exposure on Photo-Activated Activity

The effect of EGF-IRDye 700DX pre-exposure to white light versus greenlight on photo-activated cell killing was also evaluated in A431 cells.EGF-IRDye 700DX was pre-exposed to different types of light and theeffect of light treatment on photo-activated killing activity wasevaluated. Five microliters of EGF-IRDye 700DX conjugate (DAR 2) wasadded per clear HPLC vial at a EGF-IRDye 700DX concentration of 1.14mg/mL. The following conditions were tested: (1) the antibody-IRDye700DX conjugate stored at 4° C. protected from light (“4° C.”, used asthe control); (2) antibody-IRDye 700DX conjugate placed in a clear glassHPLC tube under a Halogen lamp at 2500 lux for 24 hrs (“white light”);(3) antibody-IRDye 700DX conjugate placed in a clear glass HPLC tubewrapped in aluminum foil to protect from light exposure under Halogenlamp at 2500 lux for 24 hrs (“no light”), used as a control for thermalheating effects on the formation of aggregates); and (4) antibody-IRDye700DX conjugate placed in a clear glass HPLC tube and exposed to greenLED lamp at 2500 lux for 24 hrs (“green light”).

To assess cell killing activity, A431 cells were washed two times withserum free media, and incubated in serum free media alone for one hourat 4° C. The cells were then washed one time with serum free media andincubated with serum free media alone or serum free media containing 1μg/mL of EGF-IRDye 700DX (“no light”), serum free media containing 1μg/mL of EGF-IRDye 700DX pre-exposed to white light (“2500 Lux Whitelight”), or serum free media containing 1 μg/mL EGF-IRDye 700DXpre-exposed to green light for one hour at 4° C. (“2500 Lux Greenlight”). The cells were then washed one time with serum free media.

To induce cell killing, the cells were either protected from light(light dose 0 J/cm²) or were illuminated with a 690 nm laser withdifferent light dosimetries (8 J/cm², 32 J/cm² or 64 J/cm²).

As shown in FIG. 11B, EGF-IRDye 700DX light-dependent killing activitywas sensitive to pre-exposure to white light. A431 cells incubated withEGF-IRDye 700DX that had been protected from light exposure but notthermal heating under white light from a halogen lamp at 2500 lux for 24hours exhibited light-dependent killing. A431 cells incubated withEGF-IRDye 700DX that had been exposed to white light from a halogen lampat 2500 lux for 24 hours no longer exhibited light-dependent killingactivity. A431 cells incubated with EGF-IRDye 700DX that had beenexposed to green light from a green LED lamp at 2500 lux for 24 hoursexhibited light-dependent killing activity comparable to that of the “nolight” EGF-IRDye 700DX. A431 cells incubated with serum free media alonedid not exhibit light-dependent killing activity.

B. Cholera Toxin B-IR700 Conjugate

To assess if cell killing can be mediated by a molecule that binds tonon-protein molecules, Cholera Toxin B (Catalog No: C9903-2MG, SigmaAldrich, St. Louis, Mo.) was conjugated to IRDye 700DX and evaluated toassess its killing activity upon pre-exposure to different wavelengthsof light. Cholera toxin B binds specifically to glycolipid, GM1, whichis a non-protein surface targeting molecule moiety.

1. Cholera Toxin B Conjugation

The protocol used for labeling of the Cholera Toxin B with IRDye 700DXwas substantially the same as the protocol for antibody conjugationdescribed in Example 5, except that the prior to conjugation, theCholera Toxin B solution was first exchanged with phosphate buffersaline pH 7 using a 3,000 Dalton molecular weight cutoff centrifugalfilter. For the conjugation, the solubilized IR700 NHS Ester was thenadded to the Cholera Toxin B solution at a 2 (IR700 NHS Ester) to 1(Cholera Toxin B) molar ratio. After the conjugation reaction, which wasperformed substantially as described in Example 8, the Cholera Toxin Bconjugate solution was then exchanged then exchanged with 24 mL of PBSpH 7 using a 10,000 Dalton molecular weight cutoff filter to remove freedye, glycine, and glycine-IR700, and to adjust the pH back to pH 7.

2. Cholera Toxin B-M700 Killing Activity

Photo-activated cell killing using cholera toxin B-IR700 was assessed inBxPC3 cells. BxPC3 cells were washed three times with RPMI mediasupplemented with 1% Penicillin/Streptomycin (serum free media), thenincubated with serum free media only or serum free media containing 2μg/mL of cholera toxin B-IRDye 700DX (DAR ˜2.9 per pentamer) for onehour at 4° C. The cells were then washed two times with serum freemedia.

To induce IR700-dependent killing, the cells were either protected fromlight (light dose 0 J/cm²) or were illuminated with a 690 nm laser withdifferent light dosimetries (2 J/cm², 8 J/cm² or 32 J/cm² or 96 J/cm²).Cell death was evaluated as described in Example 6 using CellToxreagent. The normalized percentage of dead cells was calculated bysubtracting all wells by the percentage of dead cells from the no lightcomplete culture media only control, dividing by cholera toxin B-IRDye700DX at 96 J/cm² minus no light complete culture media only control,and multiplied 100.

As shown in FIG. 12A, the effect of light dose on light-dependentkilling of BxPC3 cells was dose dependent, as evidenced by an increasein the normalized percent of dead BxPC3 cells that had been incubatedwith 2 μg/mL Cholera Toxin B-IRDye 700DX for 1 hour at 4° C. followed byirradiation in the presence of increasing light dose. No light dosedependent killing of BxPC3 cells treated only with complete culturemedia was observed.

To assess specificity of the photo-activated cell killing activity,BxPC3 cells were washed three times with serum free media, thenincubated with complete culture media alone or complete culture mediacontaining 100 μg/mL unconjugated cholera toxin B for one hour at 4° C.The cells were then washed one time with serum free media, and incubatedfor one hour at 4° C. with serum free media only, serum free mediacontaining 2 μg/mL of cholera toxin B-IRDye 700DX, or 100 μg/mLunconjugated cholera toxin B with 2 μg/mL of cholera toxin B-IRDye700DX. The cells were then washed two times with serum free media. Toinduce IR700-dependent killing, the cells were either protected fromlight (light dose 0 J/cm²) or were illuminated with a 690 nm laser withat 96 J/cm² and cell death was evaluated as described above.

As shown in FIG. 12B, the results showed that pre-incubation of BxPC3cells with 100x excess of the unconjugated cholera toxin B blockedCholera Toxin B-IRDye 700DX light-dependent killing in BxPC3 cells,thereby indicating that the killing activity is dependent on binding ofthe Cholera toxin B to cells.

3. Effects of Light Pre-Exposure on Cell Killing Activity

The effect cholera Toxin B-IRDye 700DX pre-exposure to white versusgreen light on photo-activated killing activity was evaluated. Tenmicroliters of Cholera Toxin B-IRDye 700DX conjugate (DAR 2.9) was addedper clear HPLC vial at a Cholera Toxin B-IRDye 700DX concentration of 1mg/mL. The following conditions were tested: (1) Cholera Toxin B-IRDye700DX conjugate placed in a clear glass HPLC tube wrapped in aluminumfoil to protect from light exposure under Halogen lamp at 2500 lux for24 hrs (“no light”, used as a control for thermal heating effects on theformation of aggregates); (2) Cholera Toxin B-IRDye 700DX conjugate wasplaced in a clear glass HPLC tube under a Halogen lamp at 2500 lux for24 hrs (“white light”); or (3) Cholera Toxin B-IRDye 700DX conjugate wasplaced in a clear glass HPLC tube and exposed to green LED lamp at 2500lux for 24 hrs (“green light”).

Cell killing induced by the cholera Toxin B-IRDye 700DX pre-exposedunder the various conditions was assessed on BxPC3 cells as describedabove. Thus, all BxPC3 cell treatments were incubated with either serumfree media alone or serum free media containing Cholera Toxin B-IRDye700DX that had undergone pre-exposure to light of different wavelengthsof light as described above.

As shown in FIG. 12C, light-dependent killing activity mediated byCholera Toxin B-IRDye 700DX was sensitive to pre-exposure to whitelight. BxPC3 cells incubated with Cholera Toxin B-IRDye 700DX that hadbeen protected from light exposure but not thermal heating under whitelight from a halogen lamp at 2500 lux for 24 hours exhibitedlight-dependent killing. BxPC3 cells incubated with Cholera ToxinB-IRDye 700DX that had been exposed to white light from a halogen lampat 2500 lux for 24 hours no longer exhibited light-dependent killingactivity. BxPC3 cells incubated with Cholera Toxin B-IRDye 700DX thathad been exposed to green light from a green LED lamp at 2500 lux for 24hours exhibited a slight decrease in light-dependent killing activity,but substantially less than that of the white light exposed CholeraToxin B-IRDye 700DX treated cells. BxPC3 cells incubated with serum freemedia alone did not exhibit light-dependent killing activity.

C. Influenza Virus-IR700

The following studies were performed to assess whether virus particlescan be conjugated with phthalocyanine dyes such as IRDye 700DX forphoto-activated cell killing. Effect of pre-exposure to white light onphoto-activated virus-IR700 conjugate killing was also assessed.

1. Influenza Virus (X-31) Conjugation

Frozen solid aliquots of IRDye 700DX NHS Ester (Cat. No. 929-70011;Li-COR, Lincoln, Nebr.) were thawed at room temperature, then dissolvedwith DMSO to achieve a 10 mg/mL concentration. In a dark environment, 10μg of IRDye 700DX NHS Ester was added to a 65,536 HA titer units ofInfluenza A X-31, A/Aichi/68 (H3N2) stock (Catalog No: 10100375, CharlesRiver Laboratories, Wilmington, Mass.), and placed on the lowest settingpossible on a table top vortexer for 2 hours at 25° C. A gravity flowcolumn was used to separate the virus conjugate from the free dye byloading 100 μL of virus solution to a pre-phosphate buffer salineequilibrated Nap 5 gravity flow column (Catalog No: 17-0853-02, GEHealthcare Life Sciences, Pittsburgh, Pa.). After adding 650 μL ofphosphate buffer saline, the flow through was discarded. An additional400 μL phosphate buffer saline was loaded to the column and the flowthrough, which contained the conjugated virus, was collected. Prior tousing the virus for experiments, the virus conjugate solution wasfiltered with a 0.2 μm pore size PVDF filter to remove any insolubleaggregates.

2. Influenza Virus (X-31)-IRDye 700DX Killing Activity

Vero cells were incubated with influenza virus (X-31)-IR700 to assess ifcells associated with the influenza virus (X-31)-IR700 were susceptibleto killing after light irradiation. Vero cells were washed four timeswith EMEM media supplemented with 1% Penicillin/Streptomycin (serum freemedia). Virus inoculation media was made by mixing 1200 μL serum freemedia with 400 μL of purified influenza virus (X-31)-IRDye 700DX flowthrough (prepared as described above), which was then filtered with a0.2 μm pore size PVDF filter to remove any aggregates. 100 μL of virusinoculation media or 100 μL of serum free culture media was added to thecells, and incubated for 1 hr at 4° C. The cells then were washed oncewith 100 μL of serum free media.

Virus-associated cells or control Vero cells were then either protectedfrom light (light dose 0 J/cm²) or were illuminated with a 690 nm laserwith different light dosimetries (2 J/cm², 8 J/cm², 32 J/cm² or 96J/cm²). Cell death was evaluated as described in Example 6 using CellToxGreen.

As shown in FIG. 13A, Vero cells that were inoculated with Influenzavirus (X-31)-IRDye 700DX were killed in a light dose-dependent manner.Vero cells incubated in complete culture media without virus did notexhibit light dependent killing.

3. Effects of Light Pre-Exposure on Conjugate Activity

The influenza virus (X-31)-IRDye 700DX was tested for the effect ofpre-exposure to light on photo-activated light-dependent killingactivity under three different light-exposure conditions, including tothe different wavelengths of white light vs. green light. Approximately130 uL of influenza virus (X-31)-IRDye 700DX flow through was added perclear HPLC vial and tested after exposure to the following conditions:(1) influenza virus (X-31)-IRDye 700DX conjugate was placed in a clearglass HPLC tube wrapped in aluminum foil to protect from light exposureunder a Halogen lamp (Catalog No: PL-800, Dolan-Jenner, Boxborough,Mass.) at 2500 lux for 18 hrs (“no light”, to control for thermalheating effects); (2) the influenza virus (X-31)-IRDye 700DX conjugatewas placed in a clear glass HPLC tube under a halogen lamp at 2500 luxfor 18 hrs (“white light”); (3) influenza virus (X-31)-IRDye 700DXconjugate was placed in a clear glass HPLC tube and exposed to green LEDlamp (Catalog No: Green-ECS GP19 EcoSmart) at 2500 lux for 18 hrs((“green light”).

Cell killing induced by inoculation of Vero cells with influenza virus(X-31)-IRDye 700DX pre-exposed under the various conditions was assessedas described above after illumination with a 690 nm laser with a lightdose of 96 J/cm². Thus, all Vero cell treatments were incubated witheither serum free media alone or serum free media containing influenzavirus (X-31)-IRDye 700DX that had undergone pre-exposure to light ofdifferent wavelengths of light.

As shown in FIG. 13B, light-dependent killing activity mediated byinfluenza virus (X-31)-IRDye 700DX is sensitive to pre-exposure to whitelight. Vero cells incubated with influenza virus (X-31)-IRDye 700DX thathad been protected from light exposure with aluminum foil (“no light”)exhibited light-dependent killing. However, the extent of cell killingwas decreased in Vero cells incubated with influenza virus (X-31)-IRDye700DX that had been exposed to white light from a halogen lamp at 2500lux for 18 hours compared to cell treated with the “no light” influenzavirus (X-31)-IRDye 700DX that had been protected from light. Incontrast, incubation of Vero cells with influenza virus (X-31)-IRDye700DX that had been exposed to green light from a green LED lamp at 2500lux for 18 hours exhibited the same photo-activated killing activity asthat of the “no light” influenza virus (X-31)-IRDye 700DX that had beenprotected from light. Vero cells incubated with serum free media alonedid not exhibit light-dependent killing activity.

Example 11: Assessment of Cell Killing Activity of AdditionalMolecule:IR700 DX Conjugates

Studies were performed to assess the cell killing activity of additionalnon-antibody IR700 conjugates that can bind to non-protein surfacemolecules. In an exemplary additional study, the effect of Sambucusnigra Lectin (SNA; also called Elderberry lectin, EBL) (Catalog No:L-1300, Vector Labs, Burlingame, Calif.) conjugated to IRDye 700DX wasevaluated to assess its killing activity. SNA binds specifically toalpha(2,6)-linked sialic acids on glycoproteins on cells. The SNA-IR700also was assessed for light-induced aggregation using size exclusionchromatography, but in this exemplary experiment there was no effect onthe size exclusion chromatography of the SNA-IR700 conjugate exposed towhite light versus green light.

1. Elderberry Lectin (SNA) Conjugation

The protocol used for labeling of the SNA with IRDye 700DX issubstantially the same as the protocol for antibody conjugationdescribed in Example 5.

2. SNA-IR700-Light-Dependent Killing Activity

To assess if SNA-IR700 was able to elicit cell killing after lightirradiation, cell killing was assessed in BxPC3 cells. BxPC3 cells weredissociated from the cell culture plate and the cell culture mediacontaining RPMI media supplemented with 10% Fetal Bovine Serum and 1%Penicillin/Streptomycin (complete culture media) was exchanged for RPMImedia supplemented with 1% BSA and 1% Penicillin/Streptomycin (bindingmedia). The BxPC3 cells were transferred to separate tubes containingbinding media only or binding media containing 10 μg/mL SNA-IRDye 700DXat a dye antibody ratio (DAR) of ˜2.5), and incubated for one hour at 4°C. The cells were then transferred to plates pre-coated with 200 μg/mLunconjugated SNA (1 hr coating treatment at 37° C., and washed 3 timeswith serum free media) to block non-specific binding of the SNA-IRDye700DX to the plates.

To induce IR700-dependent killing, the cells were either protected fromlight (light dose 0 J/cm²) or were illuminated with a 690 nm laser withdifferent light dosimetries (8 J/cm⁻², 32 J/cm⁻² or 96 J/cm²). Celldeath was evaluated as described in Example 6 using CellTox Green.

As shown in FIG. 14A, BxPC3 cells incubated with SNA-IRDye 700DXexhibited light dependent killing. BxPC3 cells treated with completeculture media in the absence of an IR700 conjugate did not exhibit lightdependent killing.

To assess the specificity of the cell killing, BxPC3 cells were treatedwith sialidase A, which cleaves alpha(2,6)-linked sialic acids, thereceptor for SNA. BxPC3 cells were dissociated from the tissue cultureflask, and fixed with 10% formalin for 20 minutes. The cells were thenwashed 3 times with PBS, and treated with a 1× reaction buffer alone(diluted from a 5× Glyco Sialidase A-51 reaction buffer, catalog numberGK80045, Prozyme), 1× reaction buffer containing 0.025 U sialidase A, or1× reaction buffer containing 0.075 U sialidase A for 2 hours at 37° C.The cells were then washed three times with PBS, and then incubated withPBS alone or PBS containing 10 μg/mL SNA-IRDye 700DX for 1 hour at 4° C.

After the incubation, the cells were washed three times with PBS,stained with DAPI nuclear stain, and then plated onto 96 well dish andimaged on an epi-illumination fluorescent microscope. At least 10regions were chosen and imaged to detect DAPI nuclear stain andSNA-IRDye 700DX fluorescent signal. To compare fluorescent intensity ofthe tested groups, background subtraction was performed by subtractingthe minimum pixel intensity of a given image from all other pixels inthe same image. The DAPI nuclear signal was thresholded and used as therepresentative area for each cell. The segmented DAPI image was thenused to determine the area for each individual cell to be quantified foraverage fluorescence intensity in the channel used to image theSNA-IRDye 700DX. Because the SNA-IRDye 700DX staining is a membranestain that is diffuse and because an epi-illumination microscope wasused, the average fluorescent signal measured from the masked region asdefined by the DAPI nuclear stain could be used as a representativeaverage fluorescent intensity for SNA-IRDye 700DX staining per cell. Theaverage fluorescence intensity was collect for hundreds of cells pertreatment condition and plotted in a box and whisker plot.

The fluorescent intensity results of the tested groups after treatmentof cells with sialidase A is shown in FIG. 14B. The results showed thata dose dependent increase in sialidase A treatment resulted in aconcomitant decrease in SNA-IRDye 700DX staining in the sample. Dosedependent increase in sialidase A treatment did not result in any changein fluorescence from the channel used to detect the SNA-IRDye 700DX whenBxPC3 cells were not stained with SNA-IRDye 700DX.

Example 12: IR700-Conjugate-Mediated PIT Killing of Bacterial Pathogens

The following studies were performed to assess whether antibodiesdirectly conjugated to a phthalocyanine photosensitizer such as IRDye700DX can kill bacterial cells by binding to proteins displayed on itscell surface. Protein A is a protein displayed on the cell surface ofStaphylococcus aureus (S. aureus) that binds to the Fc region ofantibodies.

Cetuximab-IR700, conjugated substantially as described in Example 1, wasused in these studies. S. aureus was acquired from American Type CultureCollection (ATCC) ID 6538. S. aureus was grown on either Brain HeartInfusion (BHI) agar plates for colony selection and counting, or BHIbroth (complete culture media) for population expansion.

To evaluate bacterial cell-induced PIT killing, S. aureus was incubatedwith 100 μg/mL of cetuximab-IRDye 700DX for one hour at roomtemperature. The cells were then illuminated with a 690 nm laser at 0 or300 J/cm⁻². The number of remaining viable bacterial cells wasdetermined by counting colony forming units (CFU) on BHI agar platesunder the following conditions. As a control, the number of viablebacterial cells also was assessed in cells treated with cetuximab-IRDye700DX incubation alone but without laser illumination, laserillumination alone, or untreated. Percent of viable CFU was normalizedto bacterial cells with no treatment.

The results are shown in FIG. 15, which shows that PIT-mediated cellkilling of S. aureus can occur in the presence of an antibody-IR700conjugate that binds to Protein A. Only the bacterial cells that wereincubated with cetuximab-IRDye 700DX with subsequent laser illuminationhad a statistically significant CFU reduction in comparison to the otherthree groups.

Example 13: IR700-Conjugate-Mediated PIT Killing of Virus Pathogens

The following studies were performed to assess whether virus infectivitycan be inhibited by performing PIT on virus particles withphthalocyanine-labeled anti-virus antibodies. An exemplary study wasperformed using influenza virus as a specific example in which indirectPIT treatment was performed against influenza virus particles coatedwith mouse anti-influenza virus A (H3N2) and goat anti-mouse Fab-IRDye700DX antibodies. Because indirect labeling of primary unconjugatedantibodies with secondary antibody-IRDye 700DX conjugates can induce PITkilling similar to that of direct conjugated primary antibodies, thefindings can be generalized to directly conjugated anti-virus-IRDye700DX antibodies. Thus, these results demonstrate that PIT treatment canlead to inhibition of virus infection

AffiniPure Fab Fragment Goat anti-mouse IgG1 specific (GtxMs Fab)antibody (Catalog number: 115-007-185, Jackson ImmunoResearchLaboratories, West Grove, Pa.) was conjugated to IR700 substantially asdescribed in Example 5, except the GtxMs Fab antibody solution was firstexchanged with phosphate buffer saline pH 7 using a 10,000 Daltonmolecular weight cutoff centrifugal filter, then the antibody solutionpH was adjusted to a pH of 8.5 with addition of phosphate buffer atpH=9. Frozen solid aliquots of IRDye 700DX NHS Ester (Cat. No.929-70011; Li-COR, Lincoln, Nebr.) were thawed at room temperature, thendissolved with DMSO to achieve a 10 mg/mL concentration. In a darkenvironment, the solubilized IR700 NHS Ester was then added to theantibody solution at a 2 (IR700 NHS Ester) to 1 (antibody) molar ratio.The conjugation reaction proceeded at 25° C. for 2 hours protected fromlight. Glycine (pH 8.2) was added to a final concentration of 10 mM for15 minutes to quench the reaction. The antibody conjugate solution wasthen exchanged with 24 mL of PBS pH 7 using a 10,000 Dalton molecularweight cutoff centrifugal filter to remove free dye, glycine, andglycine-IR700, and to adjust the pH back to pH 7.

For PIT, influenza A virus was indirectly associated with IR700 bymixing 1 μg of mouse Anti-Human Influenza A (H3N2) (F49) (Catalog No:M146, TaKaRa, Katsushika Tokyo, Japan) and 1 μg of GtxMs Fab-IRDye 700DXfor 5 minutes at 25° C. in the dark, followed by a 30 minute incubationwith 16,384 HA titer units of Influenza A X-31, A/Aichi/68 (H3N2) stock(Catalog No: 10100375, Charles River Laboratories, Wilmington, Mass.)for 30 minutes at 25° C. in the dark. Approximately 875 μL of EMEMsupplemented with 1% Penicillin/Streptomycin (serum free media) wasadded, and the incubated virus was filtered with a 0.2 μm pore size PVDFfilter to remove any insoluble aggregates (virus inoculation media). Theincubation was performed in duplicate. For one of the duplicate samples,the antibody-virus solution was exposed 144 J/cm⁻² of 690 nm light,while the other sample was protected from light.

PIT-treated virus were evaluated for infectivity with Vero cells. Twentyfour hours prior to labeling influenza virus (X-31) with themouse-anti-influenza virus A (H3N2) and the GtxMs Fab-IRDye 700DX,125,000 Vero cells were plated in a 6 well dish. The following day afterseeding the cells and after labeling the influenza virus (X31) with themouse anti-influenza virus (H3N2) antibody with GtxMs Fab-IRDye 700DX,the cells were washed four times with serum free media. The cells werethen incubated with 100 μL of light-treated virus inoculation media, nolight treated virus inoculation media, or serum free media for 1 hour at37° C. The media was then replaced with EMEM supplemented with 0.3%bovine serum albumin (BSA) and 1% Penicillin/Streptomycin. After 14hours post virus inoculation, the cells were trypsinized, andresuspended in EMEM supplemented with 10% fetal bovine serum and 1%Penicillin/Streptomycin, and placed into Eppendorf tubes. Cells werethen fixed with 10% formalin for 20 minutes, and subsequently washed 3times with phosphate buffer saline (PBS, pH 7). For each wash step,cells were spun down at 1500 rpm for 3 minutes, supernatant was removed,and the cell pellet was resuspended with 1 mL of PBS.

The cells were then incubated for 30 minutes at 25° C. with “blockbuffer” containing PBS supplemented with 3% Bovine Serum Albumin(IgG-Free, Protease-Free) (Catalog No: 001-000-162, JacksonImmunoResearch Laboratories, Wilmington, Mass.) and 0.08% saponin. Thecells were then incubated for 1 hour 10 minutes at 25° C. with 1:2000mouse (IgG2a) Anti-Influenza A Virus Nucleoprotein antibody [AA5H](Catalog no: ab20343, Abcam, Cambridge, United Kingdom) diluted in blockbuffer. The cells were subsequently washed 3 times with block buffer byspinning the cells down at 1500 rpm for 3 minutes, removing thesupernatant, resuspending the cell pellet with 100 μL of block buffer,and incubating the cells for at least 5 minutes at 25° C. prior to thenext wash. After washing out the primary antibody, the cells wereincubated for 30 minutes at 25° C. with 1:250 AlexaFluor 488-conjugatedAffiniPure Goat Anti-Mouse IgG FcGamma Subclass 2a specific (Catalog No:115-545-206, Jackson ImmunoResearch Laboratories, Wilmington, Mass.)diluted in block buffer. The cells were then washed 3 times with 100 μLper wash of block buffer with at least 5 minutes per wash step, followedby 3 additional washes with PBS. The cells were then spotted on a 96well plate, and imaged with a fluorescent microscope (Evos, LifeTechnologies). At least 12 random regions of interest were randomlychosen to obtain the brightfield image and corresponding fluorescentimage taken with the GFP excitation and emission cube. The brightfieldimage was used to obtain the total number of cells, and the fluorescentimage was used to detect the presence of nucleoprotein expression, areadout that the cell was infected with influenza virus infection.

The effect of influenza virus particles coated with anti-HA and goatanti-mouse IRDye 700DX (GtxMs Fab-IRDye 700DX) exposure to 690 nm lighton virus infectivity was evaluated. The results in FIG. 16 show that PITon influenza virus particles using pre-complexed mouse anti-influenzavirus (H3N2) with GtxMs Fab-IRDye 700DX abrogates influenza virusinfection. Vero cells incubated with virus coated with pre-complexedmouse anti-influenza virus (H3N2) and GtxMs Fab-IRDye 700DX that werenot exposed to 690 nm light resulted in robust virus infection, withabout 97.4% of the Vero cells staining for influenza virus nucleoproteinexpression. In stark contrast, Vero cells incubated with PIT-treatedvirus coated with mouse anti-influenza virus (H3N2) and GtxMs Fab-IRDye700DX exhibited a significant decrease in virus infection, with only1.8% of the cells staining for influenza virus nucleoprotein expression.

Example 14: IR700-Conjugate-Mediated PIT Killing of Pathogen InfectedCells

The following studies were performed to assess whether virus-infectedcells can be selectively treated with PIT with anti-virus antibodieslabeled with phthalocyanine dyes (such as IRDye 700DX) either throughdirect conjugation or indirect labeling with secondary antibodyconjugates. The exemplary data includes performing PIT on influenzavirus-infected cells using indirect PIT with mouse anti-influenza virus(H3N2) antibodies and Goat anti-mouse-IRDye 700DX secondary antibodies.

In this study, conjugation of AffiniPure Fab Fragment Goat anti-mouseIgG1 specific (GtxMs Fab) antibody with IR700 was performedsubstantially as described in Example 5.

Vero cells were infected with Influenza virus prior to treating thevirus or cells with PIT. Approximately 5,000 Vero cells were plated in a96 well clear bottom, black plates. The following day after seeding thecells, the cells were washed four times with 100 μL of EMEM supplementedwith 1% Penicillin/Streptomycin (serum free media), then incubated withserum free media containing 327.68 HA titer units of Influenza A X-31,A/Aichi/68 (H3N2) (Catalog No: 10100375, Charles River Laboratories,Wilmington, Mass.) per well. The cells were then incubated with thevirus inoculation media or serum free media for 90 minutes at 37° C.,after which the virus inoculation media was then replaced with EMEMsupplemented with 0.3% bovine serum albumin (BSA) and 1%Penicillin/Streptomycin.

Virus infected cells were then labeled with IR700 14 hours post virusinoculation. Briefly, the cells were incubated for one hour at 4° C.with 1 μg/mL of mouse Anti-Human Influenza A (H3N2) (F49) (Catalog No:M146, TaKaRa, Katsushika Tokyo, Japan) diluted with EMEM supplementedwith 10% fetal bovine serum and 1% Penicillin/Streptomycin (completeculture media). The cells were then washed one time with completeculture media, and then incubated for one hour at 4° C. with 5 μg/mL ofGtxMs-IRDye 700DX diluted in complete culture media. The cells were thenwashed once with 100 μL of complete culture media. To induce PIT, thecells were illuminated with a 690 nm laser at 64 J/cm⁻² or protectedfrom light (“no light”).

Cell death was evaluated using CellTox Green reagent. After the lighttreatment, all cells were incubated with 1× CellTox Green reagentdiluted in complete culture media for 15 minutes at 37° C., then imagedwith a fluorescent microscope (Evos, Life Technologies). At least 5random regions of interest per well for at least three different wellswere randomly chosen to obtain the brightfield image, anti-influenzavirus fluorescent image using a Cy5 excitation and emission cube, andCellTox Green fluorescent image using a GFP excitation and emissioncube. Cells that were then scored for anti-influenza virus staining asan indication for the cell being virus infected. Of the virus infectedcells, the cells were then scored for whether there was CellTox Greenstaining.

As shown in FIG. 17, the results showed that PIT induced cell death wasobserved in influenza virus-infected Vero cells that had beensequentially labeled with mouse anti-influenza virus (H3N2) and goatanti-mouse IRDye 700DX (GtxMs-IRDye 700DX) followed by lightirradiation. The extent of cell death that was observed waslight-dependent, since only negligible cell death was observed in theabsence of light treatment.

Example 15: IR700-Conjugate-Mediated PIT Killing of Neurons

The following study was performed to assess whether neurons can bekilled by PIT using conjugates of IRDye 700DX. Dorsal Root Ganglion(DRG) neurons were subjected to PIT with the B subunit of Cholera Toxinconjugated with IRDye 700 DX. Irradiation with laser light of 690 nmresulted in complete cell death as measured in a luminescence based celltoxicity assay. Without light administration no significant cell deathwas observed. The findings demonstrate that PIT can be an effectivetreatment to kill neurons, and more broadly, to kill non-cancer cells,including primary cells.

Rat embryonic DRGs were obtained from Lonza (catalog number R-eDRG-515,Lonza Walkersville, Inc., Walkersville, Md.) in cryo-preserved formatand stored in liquid Nitrogen until used. Black-wall 96-well plates werecoated with 50 μL PBS per well containing 30 μg/mL poly-D-lysine(Sigma-Aldrich, catalog P0899, St. Louis, Mo.) and 2 μg/mL laminin(Sigma-Aldrich, L2020, St. Louis, Mo.) for 1 hour at room temperature,following stock solution preparation and procedures by Lonza. Thecoating solution was aspirated and the plates let dry for an hour (openlid in biosafety cabinet) and used immediately for cell seeding. Theinstructions provided by Lonza were strictly followed for thawing andplating the cells. The culture medium was PNBM supplemented at all timeswith 2 mM L-glutamine, 50 μg/mL Gentamicin, 37 ng/mL Amphotericin and 2%NSF-1, but the latter was added fresh each time before use. Thesecomponents were part of a kit (catalog number CC-4461, Lonza, Basel,Switzerland). Additionally, nerve growth factor (NGF, catalog numberN2513, Sigma, St. Louis, Mo.) was also added fresh at the time of use to100 ng/mL. To plate cells, a 0.25 mL vial was thawed and dropwisediluted with 3.75 mL culture medium, and 200 μL suspension was seededinto wells. Cells were incubated for 4 hours at 37° C. and 5% CO2, andthe medium was replaced with medium also containing the mitoticinhibitors 5-fluoro-2′-deoxyuridine (7.5 μg/mL final concentration,catalog number F-0503, Sigma, St. Louis, Mo.) and uridine (17.5 μg/mLfinal concentration, catalog number U-3003, Sigma, St. Louis, Mo.) thatwere added just before use. The medium was changed again every 3-4 days.

The conjugation of Cholera Toxin B with IR700 was performed as describedin Example 10.B.

After culturing rat embryonic DRGs for 11 days, 1 μg/mL stock solutionof Cholera Toxin B-IR700 was diluted to 40 μg/mL with culture medium and5 μL of the diluted conjugate was added directly to the wells of a96-well plate containing DRG neurons in 200 μL medium, to achieve afinal concentration of 5 μg/mL conjugate. Cells were incubated for 1hour at 37° C. The culture medium was removed, the cells washed oncewith culture medium, and 100 μL fresh culture medium was added. Thestained neurons were then illuminated with a 690 nm laser at a lightdose of 64 J/cm² (150 mW/cm²), or left protected from light as a control(“no light”).

The effect of PIT on DRG neurons was measured with the luminescencebased toxicity assay CytoTox Glo (catalog number G9291, Promega,Madison, Wis.). This assay is based on membrane integrity and employs apro-substrate for luciferase that cannot penetrate intact cells. Whencells die, damage in the plasma membrane allows enzymes to diffuse outof cells and activating the pro-substrate, now becoming a real substratefor luciferase, resulting in a luminescence signal. Plates wereequilibrated to room temperature for 15 minutes, and 50 μL assay reagentwas added. After incubating for 20 minutes at room temperature,luminescence was read on a multi-mode reader. To determine complete celldeath, 50 μL digitonin solution was added to kill remaining viablecells, and after 20 minutes luminescence was read again. The backgroundvalues from wells without cells were subtracted from each read, andpercent cell death was calculated as the ratio between luminescencebefore and after lysis with digitonin, multiplied by 100.

As shown in FIG. 18, PIT induced cell death in Rat Embryonic DRGNeurons. Irradiation with 690 nm laser light of 64 J/cm²) lead to 100percent cell death after 3 hours (left bar), whereas light protectedcells (“No Light”) remained unharmed (6% dead cells, right bar).

Example 16: Combination Treatment with Interferon Gamma andAnti-PD-L1-IR700 PIT

The following studies were performed to assess whether PIT can becombined with immune modulatory agents—which can also affect cancercells—to enhance PIT-killing activity.

A. Effect of Interferon Gamma on Cell Death

BxPC3 cells (#CRL-1687, ATCC, Manassas Va.) were seeded in 96 wellblack, clear-flat bottom dishes at 5000 cells per well, and placed in at37° C., 5% CO₂ incubator. The following day, the cells were washed oncewith RPMI 1640 supplemented with 10% FBS and 1% Penicillin/Streptomyocin(complete culture media). The cells were then incubated for 18 hourswith complete culture media containing different concentrations ofrecombinant human Interferon Gamma (IFNgamma) (carrier free) (Cat No:570202, BioLegend, San Diego, Calif.) ranging from 0 ng/mL to 3.75μg/mL.

After 18 hours, the media containing different concentrations ofinterferon gamma was replaced with complete culture media containing 1×CellTox Green (Cat No: G8731, Promega, Madison, Wis.). Wells that didnot include any cells were also incubated with 1× CellTox Green reagentdiluted in complete culture media to serve as background subtractionwells during fluorescent signal detection. The CellTox Greenfluorescence signal was measured at 24.5 hours after light treatmentusing a fluorescence plate reader. The cells were then lysed withdetergent, incubated at 37° C. for 30 minutes, and the CellTox Greenfluorescence signal was measured again post lysis. The percent deadcells was calculated by taking the ratio between background (1× CellToxGreen in complete culture media without cells) subtracted CellTox Greensignal per well prior to and post lysis and multiplying the ratio by100.

The results in FIG. 19A show the increasing IFNgamma concentrationresults in a dose-dependent increase in cell death of BxPC3 cells.

B. Effect of Interferon Gamma on PD-L1 Expression

BxPC3 cells were seeded in 12 well dishes at 145,000 cells per well, andplaced at 37° C. in a 5% CO₂ incubator. The following day, the cellswere washed once with RPMI 1640 supplemented with 10% FBS and 1%Penicillin/Streptomyocin (complete culture media). The cells were thenincubated for 18 hours with complete culture media alone, completeculture media containing 375 pg/mL of recombinant human Interferon Gamma(carrier free) (Cat No: 570202, BioLegend, San Diego, Calif.), orcomplete culture media containing 37.5 ng/mL recombinant humanInterferon Gamma (carrier free). After the 18 hour incubation with orwithout recombinant interferon gamma, the BxPC3 cells were washed onetime with complete culture media.

The cells were then incubated for one hour at 37° C. with completeculture media alone or complete culture media containing 10 μg/mLanti-PD-L1-IRDye 700DX, which was prepared as described in Example 5.

After the one hour incubation, the cells were washed three times withphosphate buffer saline (pH 7) and incubated with enzyme free celldissociation buffer (Catalog No: S-014-C, EMD Millipore, Billerica,Mass.) until cells were detached. After the cells detached, phosphatebuffer saline containing 0.5% bovine serum albumin fraction V (CatalogNo: 15260-037, ThermoFisher Scientific, Waltham, Mass.) was added to thecells, and the samples were immediately analyzed by flow cytometry forPD-L1 expression based on the fluorescent signal from the IR700 dye ofthe anti-PD-L1-IRDye 700DX. The fold increase in expression wascalculated by first subtracting the fluorescent intensity from theanti-PD-L1-IRDye 700DX staining for each treatment from the unstainedcells samples, then normalizing each treatment by subtracting thebackground fluorescent intensity as determined from the mean of the nointerferon gamma treated, anti-PD-L1-IRDye 700DX stained samples.

As shown in FIG. 19B, the results showed that increasing IFNgammaconcentration resulted in a dose-dependent increase in PD-L1 expressionin BxPC3 cells.

C. Combination of Interferon Gamma and Anti-PD-L1-IR700 Conjugate on PITCell Killing

Studies were performed to assess if treatment of cells with interferongamma to increase expression of PD-L1 can enhance anti-PD-L1-mediatedPIT killing, BxPC3 cells were seeded in 96 well white, clear-flat bottomdishes at 5000 cells per well, and placed in a 37° C., 5% CO₂ incubator.The following day, the cells were washed once with RPMI 1640supplemented with 10% FBS and 1% Penicillin/Streptomyocin (completeculture media). The cells were then incubated for 18 hours with completeculture media alone, complete culture media containing 375 pg/mL ofrecombinant human Interferon Gamma (carrier free) (Cat No: 570202,BioLegend, San Diego, Calif.), or complete culture media containing 37.5ng/mL recombinant human Interferon Gamma (carrier free).

After the 18 hour incubation with or without recombinant interferongamma, the BxPC3 cells were washed one time with complete culture media.The cells were then incubated for one hour at 37° C. with completeculture media alone or complete culture media containing 10 μg/mLanti-PD-L1-IRDye 700DX or 10 μg/mL anti-PD-L1-IRDye 700DX with 100 ug/mLunconjugated anti-PD-L1. After the one hour incubation, the cells werewashed one time complete culture media.

The cells were then illuminated with a 690 nm laser with either 96 J/cm²of light with a 690 nm laser or were protected from light (“no light”).Cell death was assessed using CellTox Green reagent as described inExample 6.

As shown in FIG. 19C, combination treatment with IFNgamma prior totreatment with the anti-PD-L1-IR700 conjugate enhanced the anti-PD-L1photo-activated killing when compared to that of anti-PD-L1-IR700 PITtreatment alone. BxPC3 cells that were not treated with interferon gammaprior to anti-PD-L1-IR700 incubation exhibited a modest increase in celldeath upon 690 nm light illumination when compared to that of the nolight control. BxPC3 cells incubated with interferon gamma, followed byincubation with anti-PD-L1-IR700 conjugate exhibited an IFNgamma dosedependent increase in basal cell death in the no light treated cells,which is consistent with the effect of IFNgamma to mediate cell death.BxPC3 cells incubated with IFNgamma, incubated with anti-PD-L1-IR700conjugate, and illuminated with 690 nm light exhibited an IFNgamma dosedependent increase in cell death relative to the no light control foreach respective treatment group. The results showed thatanti-PD-L1-IR700 PIT killing activity was specific because out-competinganti-PD-L1-IR700 binding with 10x molar excess of unconjugatedanti-PD-L1 abrogated the photo-activated killing of the anti-PD-L1-IR700conjugate as demonstrated by the same percentage of cell death in thelight and no light treatments.

The results demonstrated that combination treatment with interferongamma, an anti-cancer agent and immune modulator, and anti-PD-L1-IR700PIT exhibits enhanced anticancer activity that of anti-PD-L1-IR700 PITtreatment alone or interferon gamma treatment alone.

Example 17: Immunogenic Cell Death and Immune Activation byAntibody-IR700 Conjugate-Mediated PIT

The following studies were performed to assess whether immunestimulatory changes occur in PIT-treated cells and whether PIT-treatedcells have the potential to activate immune cells. To evaluate whatimmune stimulatory changes occur in PIT-treated cells, cancer cellstreated with and without PIT were evaluated for expression ofimmunogenic cell death (ICD) markers. Immunogenic cell death is aspecific type of cell death exhibited by necrotic cells, and ischaracterized by increased presentation and release of immunestimulatory markers. Cells exhibiting ICD display membrane changes suchas elevated surface expression of heat shock protein 90, and secretionof soluble, intracellular markers known as danger associated molecularpatterns (DAMPs), such as ATP and high-mobility group-box protein(HMGB1) (Kromer et al. (2013) Annual Review of Immunology, 31:51-72). Asshown below, PIT-treated cancer cells exhibit increased HMGB1 secretionwhen compared to that of the non-PIT treated cells.

Because the PIT-treated cells exhibited elevated release of HMGB1,follow-up studies were performed to evaluate whether PIT-treated cellscould activate immune cells. To determine whether the immune cells couldbe activated by PIT-treated tumor cells, the PIT and non-PIT treatedcancer cells were co-cultured with monocyte derived immature dendriticcells (iDCs). The surface expression of DC maturation/activation markersCD80, CD86, CD40 and MHCII, which get upregulated upon inflammatorystimuli such as immunogenic cell death via PIT, were observed for anychanges. Enhancement of co-stimulatory molecules CD80, CD86 and CD40indicates augmentation in the ability of DCs to activate T cells andincreased MHCII represents increased antigen presentation capabilitiesas DCs mature. Increased expression of both costimulatory molecules andMHCII was seen on iDCs exposed to tumor killed via PIT as compared tocontrol (non-PIT treated tumor cells).

A similar tumor: APC co-culture was performed using another model systemusing THP1 cells, a human monocytic cell line that is widely used for invitro based APC activation and functional assays. Upregulation ofactivation makers CD86 was seen on THP1 cells that are exposed to PITkilled tumor cells as opposed to THP1 cells which were co-cultured withnon PIT treated tumor cells further confirming the immune-stimulatorypotential of PIT.

Altogether, the data indicated that PIT-treated cells exhibit markerscharacteristic of ICD, and that the PIT-treated cells have the potentialto activate immune cells. Therefore, combination treatment with PIT withan immune-modulating agent may further enhance the immune activatingpotential of PIT.

A. Estimation of the HMGB1 Levels from Tumor Cells Subjected to PIT ViaCetuximab-IR700

A431 and FaDu tumor cell lines were grown in complete RPMI 1640 andcomplete EMEM media, respectively. The cells were plated at 15,000 cellsin 100 μL total volume per well in a 96 well tissue culture plate foradherence overnight. The viability of the cells prior to plating waschecked via trypan blue exclusion method and >95% cells were viable.

The next day the cells were treated with cetuximab-IR700 (prepared asdescribed in Example 1) at 500 ng/mL for 1 hr at 37° C. in the CO₂incubator and then irradiated with 690 nm laser at a light fluence of 32J/cm². The controls represented wells corresponding to the groups nottreated with light.

After undergoing PIT, the media was removed from the treated cellsfollowed by washing of the cells once with PBS. This was followed byaddition of serum free version of the media and incubation for 1 hr at37° C. in the CO₂ incubator. The supernatant was collected postincubation and stored at −20° C. until use.

The culture supernatants from various treated wells were subjected toHMGB1 ELISA (IBL International, cat #ST51011) as per manufacturer'sinstructions. Briefly, lyophilized HMGB1 control and standard weresolvated with diluent buffer according to kit instructions. Acalibration standard curve was prepared by diluting HMGB1 standard stock1:4 in diluent buffer, then serial diluted 1:2 for a total of 6 points(80 ng/mL-2.5 ng/mL). 100 of diluent buffer was added to each used wellof the ELISA plate provided in the kit. 10 of standard, control, orsample was added to each well, the plate was sealed, and incubatedovernight at 37° C. After 20-24 hours unbound sample was washed awaywith provided wash buffer (diluted to 1× with distilled water).Lyophilized enzyme conjugate was solvated with enzyme conjugate diluentaccording to kit instructions and was added to washed plate at 100μL/well. The plate was gently tapped to mix and was then sealed andincubated at room temperature for 2 hours. Excess enzyme conjugate wasthen washed off with 1× wash buffer and a 1:1 mix of colrea A and colreaB solutions added to plate at 100 μL/well and incubated for 30 min atroom temperature. The reaction was then stopped by adding 100 μL/well ofstop solution and gently tapping the plate to mix. The amount of yellowproduct was quantified by its absorption at 450 nm. The HMGB1 standardcurve was graphed with 4 parameter logistics and the test sample datainterpolated into the standard curve to determine HMGB1 concentration ineach sample. The data was depicted as the fold increase over respectiveno light controls.

As shown in FIG. 20A, PIT via cetuximab-IR700 resulted in a robust HMGB1secretion from the tumor cells. Both A431 and FaDu exhibited massiverelease of HMGB1 as compared to the no light controls.

B. Determination of the Upregulation of DC Maturation Markers CD80,CD86, CD40, and MHCII on DCs Co-Cultured with PIT-Treated Tumor Cells

FaDu cells were grown in complete EMEM media. The cells were plated in100 μL total volume per well in a 96 well tissue culture plate foradherence overnight. The viability of the cells prior to plating waschecked via trypan blue exclusion method and >95% cells were viable.

The next day, the cells were treated with cetuximab-IRDye 700DX at 500ng/mL for 1 hr at 37° C. in the CO₂ incubator and then were treated withlight by subjecting the cells to 690 nm laser light fluence of 12 J/cm².The controls represented wells corresponding to the groups not treatedwith light (non-PIT treated tumor cells).

For co-culture, human iDCs (Astarte Biologics) from a healthy donor weredirectly added into the wells with PIT treated tumor cells and controlwells (non-PIT treated tumor cells) at 1:1 ratio. The co-cultures werethen incubated for 48 hours at 37° C. in the CO₂ incubator. The cellswere then detached using a non-enzymatic detachment solution. Theharvested cells from various treatment conditions were then incubatedwith live/dead discrimination dye Zombie Green (BioLegend, 1:500) for 20min at room temperature followed by washing with stain buffer.

Cells were resuspended in stain buffer and human Fc blocking reagent (BDBiosciences) was then added and cells were incubated for 20 min at roomtemperature. Anti-human CD80 (BioLegend, clone 2D10), anti-human CD86(BioLegend, clone IT2.2), anti-human CD40 (BioLegend, clone 5C3),anti-human CD11c (BD, clone B-ly6) and anti-human MHCII (BioLegend,clone L243) antibodies were then added (1:20), cells incubated for 30min at room temperature. Respective isotype control staining was alsoperformed to assess the background signal. This was followed by a washand cells resuspended in stain buffer. Data was then acquired via flowcytometry (Attune® Acoustic Focusing Cytometer) under high sensitivitymode. Flow cytometry was performed using anti-human CD14 (clone 63D₃,BioLegend, San Diego, Calif.) and anti-human CD86 (clone IT2.2,BioLegend, San Diego, Calif.) antibodies, wherein were added to cells ata 1:40 dilution, and then the cells were incubated for 30 min at roomtemperature. This was followed by a wash and then the cells wereresuspended in stain buffer. Data was then acquired via flow cytometry(Attune® Acoustic Focusing Cytometer, Thermo Fisher Scientific, Waltham,Mass.) under high sensitivity mode. Appropriate gating was done whileanalyzing the data to exclude cell debris and the data was analyzed withgating performed on live events. The results described below are basedon mean fluorescence intensity (MFI) data from each group which isplotted as fold increase over the no light controls.

FIG. 20B shows the upregulation of dendritic cell (DC) maturationmarkers on iDCs co-cultured with FaDu tumors subjected to PIT viacetuximab-IRDye 700DX. Co-culture with FaDu caused increased surfaceCD80, CD86, CD40 and WWII expression on iDCs as compared to the no lightcontrols. The Y-axis represents fold increase over respective no lightcontrols

C. CD86 Expression in THP1 Cells Upon Co-Culture with PIT and Non-PITTreated Tumor Cells

A431 cell line was grown in complete RPMI and T98G, FaDu and U87 tumorcell lines were grown in complete EMEM media. The cells were plated at15,000 cells in 100 μL total volume per well in a 96 well tissue cultureplate for adherence overnight. The viability of the cells prior toplating was checked via trypan blue exclusion method and >95% cells wereviable.

The next day the cells were treated with cetuximab-IR700 at 500 ng/mLfor 1 hr at 37° C. in the CO₂ incubator and then were treated with lightby subjecting the cells to 690 nm laser light fluence of 12 J/cm². Thecontrols represented wells corresponding to the groups not treated withlight (non-PIT treated tumor cells).

THP1 cells (ATCC® TIB202™) were grown in complete RPMI. For co-culture,15,000 THP1 cells were directly added into the wells with PIT treatedtumor cells and control non PIT treated tumor cell wells. Theco-cultures were then incubated for 24 hours at 37° C. in the CO₂incubator. On the next day, the cells were then detached using anon-enzymatic detachment solution. The harvested cells from varioustreatment conditions were then resuspended in PBS only and live/deaddiscrimination dye Zombie Green (BioLegend) was added (1:500). The cellswere incubated for 20 min at room temperature followed by washing withstain buffer.

Cells were resuspended in stain buffer and human Fc blocking reagent (BDBiosciences) was then added and cells were incubated for 20 min at roomtemperature. Flow cytometry was performed using anti-human CD14 (clone63D₃, BioLegend, San Diego, Calif.) and anti-human CD86 (clone IT2.2,BioLegend, San Diego, Calif.) antibodies, wherein were added to cells ata 1:40 dilution, and then the cells were incubated for 30 min at roomtemperature. This was followed by a wash and then the cells wereresuspended in stain buffer. Data was then acquired via flow cytometry(Attune® Acoustic Focusing Cytometer, Thermo Fisher Scientific, Waltham,Mass.) under high sensitivity mode. Appropriate gating was done whileanalyzing the data to exclude cell debris and the data was analyzed withgating performed on live events. CD14 marker was used to identify theTHP1 cells. The results were based on mean fluorescence intensity (MFI)data from each group which was plotted as fold increase over the nolight controls. The data were depicted as fold increase in CD86 surfaceexpression over respective no light controls.

As shown in FIG. 20C, CD86 was upregulated on THP1 cells co-culturedwith tumors subjected to PIT via cetuximab-IR700. Co-culture with bothA431 and FaDu cells subjected to PIT caused increased surface CD86expression on THP1 cells as compared to the no light controls.

Example 18: PIT in Combination with Treatment with an Immune-ModulatorEnhances Immune Activation

Studies were performed to assess whether there is higher immuneactivation when immune cells are primed with PIT killed tumors and alsotreated with an immune-modulator. As shown in Example 17, PIT creates aninflammatory environment which leads to activation of immune cells suchas dendritic cells (DCs) and monocytes. These PIT primed cells may alsoexhibit higher potential for further activation when combined with atreatment with an immune-modulator such as Poly I:C (a synthetic doublestranded RNA analog). To test this, PIT-treated tumor cells wereco-cultured with monocyte derived immature dendritic cells (iDCs)followed by treatment with Poly I:C, and changes in the expressionlevels of DC activation markers CD80 and CD86 was then assessed.Co-culture of iDCs with non-PIT treated tumor cells was used ascontrols. Increased CD80 and CD86 expression was seen on DCs that havebeen previously exposed to an environment where the tumor is killed viaPIT versus the condition where the tumor was not treated with PIT.

FaDu cells grown in complete EMEM media were plated in 100 μL totalvolume per well in a 96 well tissue culture plate for adherenceovernight. The viability of the cells prior to plating was checked viathe trypan blue exclusion method and >95% cells were found to be viable.The next day the cells were treated with Cetuximab IRDye 700DX (500ng/mL for 1 hr at 37° C. in an CO₂ incubator). PIT cell killing wasinduced by illumination with a 690 nm laser light at a fluence of 12J/cm². The controls represented wells corresponding to the groups nottreated with light.

For co-culture, human iDCs (Astarte Biologics) from a healthy donor weredirectly added into the wells with PIT killed tumor cells and intocontrol wells (non-PIT treated tumor cells). The co-cultures were thenincubated for 48 hours at 37° C. in the CO₂ incubator. The harvested DCswere then subjected to poly I:C treatment (1 μg/mL) for overnight. Thecells were then detached using a non-enzymatic detachment solution.

The harvested cells from various treatment conditions were incubatedwith live/dead discrimination dye Zombie Green (BioLegend, 1:500) for 20min at room temperature followed by washing with stain buffer. Cellswere resuspended in stain buffer and human Fc blocking reagent (BD) wasthen added and cells were incubated for 20 min at room temperature.Anti-human CD80 (BioLegend, clone 2D10), anti-human CD86 (BioLegend,clone IT2.2), anti-human CD40 (BioLegend, clone 5C3), anti-human CD11 c(BD, clone B-ly6) and anti-human MHCII (BioLegend, clone L243)antibodies were added (1:20) and cells were incubated for 30 min at roomtemperature. Respective isotype control staining was also performed toassess the background signal. Cells were washed and resuspended in stainbuffer. Data was then acquired via flow cytometry (Attune® AcousticFocusing Cytometer) under high sensitivity mode.

Appropriate gating was performed while analyzing the data to excludecell debris, and the data was analyzed with gating performed on liveevents. The results described below are based on median fluorescenceintensity (MFI) data from each group which is plotted as fold increaseover the no light controls.

The results in FIG. 21 showed that dendritic cells (DCs) treated withPIT in combination with an immune-modulator (Poly I:C) exhibitedenhanced immune activation as compared to DCs that were not subjected toPIT treatment in combination with an immune modulator. The pre-treatmentof DCs with PIT in combination with an immune-modulator leads toincreased CD80 and CD86 expression levels compared to the no light (noPIT) controls.

Thus the data indicated that DCs exposed to an environment created byPIT are inherently more predisposed to activation via animmune-modulator. Therefore, combination treatment with PIT with animmune modulating agent may further enhance the immune activatingpotential of PIT.

Example 19: Cetuximab Dual Labeled with IRDye 700DX and AlexaFluor488,IRDye 700DX and, DyLight755, IRDye 700DX and IRDye800 CW

Similar to the studies described in Example 4, additional studies wereperformed to assess whether antibodies conjugated to the phthalocyaninephotosensitizer IRDye 700DX may also be conjugated to a secondfluorescent dye molecule. In some aspects, the dual labeling approachcan be used to permit tumor imaging capabilities while maintainingbinding to cancer cells and subsequent PIT killing activity.

A. Conjugation Methods

The water-soluble, silicon-phthalocyanine derivative, IRDye 700DX NHSester and IRDye 800CW-NHS (also called IRDye 800 herein) were obtainedfrom LI-COR Bioscience (Lincoln, Nebr.). Alexa Fluor 488-SDP wasobtained from Life Technologies (Carlsbad, Calif.). Dylight 755-NHS wasobtained from Thermo Scientific (Waltham, Mass.). Erbitux (cetuximab)was purchased from (Myoderm USA, Norristown, Pa.). Amicon® UltraCentrifugal Filter Units (Merck Millipore Ltd, Billerica, Mass.). Allother chemicals used were of reagent grade.

1. Cetuximab-IR700 (CTX700)

Cetuximab-IRDye 700DX (CTX700) conjugate used to produce all dualconjugates described below was prepared substantially as described inExample 1. Briefly, cetuximab was reacted with 4 molar equivalents ofIRDye 700DX NHS ester for 2 hours at pH=8.5, in the dark, at roomtemperature in a using through Amicon® Ultra Centrifugal Filter Units(Cat #: UFC903024, Merck-Millipore, Billerica, Mass.) for all bufferexchange and UF/DF purification steps taking care to project theconjugate at all times from any unnecessary light exposure.

The conjugate was evaluated by size exclusion chromatography, antibodyconcentration (conc.) and dye to antibody ratio (DAR) substantially asdescribed in Example 4. The results showed the following characteristicsof the conjugate: A690=96.7% monomer; Conc=1.8 mg/mL; DAR=2.4 IRDye700DX/Ab

2. CTX700-ALX488

10 mg of CTX-700 at a concentration of 2 mg/mL in 100 mM phosphatebuffer (pH=8.5) was incubated in a light protected container with 2molar equivalents of Alexa Flour 488-SDP (5 mg/mL DMSO) at roomtemperature for 2 hours. The reaction was quenched by the addition of100 μL of 1M glycine solution (pH=8). The conjugate product wasexchanged and purified using 10 reaction volumes of 10 mM phosphatebuffer saline (PBS) pH=7.1 filtered through Amicon® Ultra CentrifugalFilter Units. The conjugate was characterized by UV-VIS, SE-HPLC, PITand Dual Emission Flow Cytometry. The UV-Vis Spectrum of CTX700-ALX488is depicted in FIG. 22A.

3. CTX700-IRDye 800

10 mg of CTX-700 at a concentration of 2 mg/mL in 100 mM phosphatebuffer (pH=8.5) was incubated in a light protected container with 2molar equivalents of IRDye 800CW-NHS (5 mg/mL DMSO) at room temperaturefor 2 hours. The reaction was quenched by the addition of 100 μL of 1Mglycine solution (pH=8). The conjugate product was exchanged andpurified using 10 reaction volumes of PBS (pH=7.1) filtered throughAmicon® Ultra 15 Centrifugal Filter Units. The conjugate wascharacterized by UV-VIS, SE-HPLC, PIT and Dual Emission Flow Cytometry.The UV-Vis Spectrum of CTX700-ALX488 is depicted in FIG. 22B.

4. CTX700-Dylight 755

10 mg of CTX-700 at a concentration of 2 mg/mL in 100 mM phosphatebuffer (pH=8.5) was incubated in a light protected container with 4molar equivalents of Dylight-NHS (10 mg/mL DMSO) at room temperature for2 hours. The reaction was quenched by the addition of 100 μL of 1Mglycine solution (pH=8). The conjugate product was exchanged andpurified using 10 reaction volumes of PBS (pH=7.1) filtered throughAmicon® Ultra 15 Centrifugal Filter Units. The conjugate wascharacterized by UV-VIS, SE-HPLC, PIT and Dual Emission Flow Cytometry.The UV-Vis Spectrum of CTX700-ALX488 is depicted in FIG. 22C.

B. Characterization of Conjugates by HPLC-SEC

The conjugates were evaluated by size exclusion chromatography (SEC) ona Shodex KW-803, 8.0×300 mm SEC column using an Agilent 1100 HPLC systemequipped with an Agilent G1315A diode array detector (DAD) monitoringthe wavelengths of 280, 488, 690, 755 and 778 nm. The running buffer wasPBS flowing at a rate of 1 mL/min. The average dye to antibody ratio(DAR) for the imaging dyes were determined using 280, 488, 690, 755 and778 nm absorbance integration areas for the antibody monomer peak afterthe appropriate extinction coefficient value correction factors wereapplied as needed for each respective dye. The CTX-700 used in all thedual conjugation reactions was analyzed by this method prior toconjugation with the imaging dye to have a DAR=2.4 before conjugationand the IRDye 700DX/Ab ratio was assumed to be unchanged afterconjugation with the imaging dye. The results are shown in Table 13.

All conjugates demonstrated both the appropriate size exclusion columnretention times (8.1-8.2 min for this method) and imaging dye wavelengthappropriate absorbance's at those retention times confirming that theimaging dye was incorporated into the CTX-700 antibody conjugate at themeasured Dye to Antibody Ratio (DAR) for Dye 2 given in the table.

TABLE 13 HPLC-SEC Determined Purity and DAR Data for Dual Conjugates DAR(IRDye Sample Purity (A690) DAR (Dye2) 700DX) CTX700 96.7 NA 2.4CTX700-Alexa Fluor 97.6 1.1 2.4 488 CTX700-IRDye 800 98.8 1.2 2.4CTX700-Dylt755 98.6 1.3 2.4

C. Cell Staining with Dual CTX-IRDye 700DX Conjugates by Flow Cytometry

BxPC3 cells (grown in RPMI containing 10% FBS and 1%Penicillin/Streptomycin) were detached using HyQTase cell detachmentreagent. Cells were then checked for viability using trypan blueexclusion dye method and >99% were found to be alive. Cells were countedusing hemocytometer and re-suspended in staining buffer (PBS containing1% BSA and 0.01% sodium azide). Approximately, 45,000 cells were stainedper staining condition with individual dye conjugates along with cellsonly control to assess the background fluorescence in the respectivechannels. Approximately, 0.25 μg of the dye conjugates/100 μL stainingvolume containing 45,000 cells was used per staining condition. Cellswere then incubated with the dye for an hour at room temperature indark. The cells were washed once with staining buffer (1800 rpm, 6 min).The stained cells were finally re-suspended in 300 μL of stainingbuffer.

The cells were read using Applied Biosystems Attune Acoustic FocusingCytometer (red and blue lasers, 6 channels) in a high sensitivity mode(slower collection mode). All necessary quality control parameters forthe machine prior to running the assay were met. The cytometer had 4fluorescent channels off of 488 nm blue laser namely BL1 (blue laser1^(st) channel), BL2, BL3 and BL4. Their corresponding band pass filtersare described in parenthesis in the table above. There are 2 fluorescentchannels off of the 638 nm red laser designated RL1 (red laser 1^(st)channel) and RL2.

For analysis, the primary Forward Scatter/Side Scatter (FSC/SSC) gatewas made so as to avoid getting debris/dead cells into the analysis. Themean fluorescence intensities of the respective channels for thecorresponding dye conjugate analyzed was determined. The results areshown in Table 14. The results showed enhanced fluorescence emissionintensities of cells labeled with the dual-conjugates relative to CTX700mono-conjugate. Thus, these results demonstrate that the dual-conjugatesmay be suitable for imaging applications in addition to photoimmunotherapy (PIT).

TABLE 14 Flow Cytometry Fluorescence Emission Data Excitation Wavelength488 nm 630 nm Emission (530 nm/ (780 nm/ Wavelength 30 nm) 60 nm) FilterFluorescence Fluorescence Sample Intensity (AU) Intensity (AU) Cellsonly 95  57 CTX700-Alexa Fluor488 515* 1003  CTX-IRDye 800 97 965CTX700-IRDye 800 88 1533* CTX700 87 1107  CTX700-Dylt755 92 5311*

D. Evaluating PIT Killing Activity

BxPC3 cells (#CRL-1687, ATCC, Manassas Va., USA) were seeded in a96-well, white-walled, clear bottom, tissue culture (TC)-treatedpolystyrene plate at a concentration of 5,000 cells/well in RPMI-1640media supplemented with 10% FBS and 1% Penicillin/Streptomycin (completeculture media) and incubated overnight at 37° C. The following daycomplete culture media was replaced with fresh complete culture mediaalone or media containing either 500 ng/ml or 100 ng/mL of test agent asindicated, and the plate was incubated for one hour at 37° C.

The cells were then illuminated with a 690 nm laser at a light dose ofeither 16 J/Cm² or 32 J/cm² or protected from light (“no light”).

The effect of the different treatment regimens on cell death wasmeasured using the fluorescent stain, CellTox Green (Catalog No: G8731,Promega, Madison, Wis., USA) substantially as described in Example 6.Results were then normalized, setting cell death after incubation with500 ng/mL cetuximab-IRDye 700DX and PIT treatment with 32 J/cm² as 100%PIT activity and cell death after incubation with complete culturemedium alone and PIT treatment with 32 J/cm² as 0% PIT activity.

As shown in FIG. 23A, BxPC3 cells incubated with 100 or 500 ng/mLcetuximab conjugated to IRDye 700DX alone or dual conjugated to IRDye700DX-DyLight-755 exhibited light-dependent and concentration-dependentkilling. The dual conjugate dosed at 500 ng/mL induced at least 90% ofthe cell killing of the cetuximab-IRDye 700DX solo conjugate controlafter PIT with 32 J/cm². When the concentration of conjugate was reducedto 100 ng/mL, cetuximab-IRDye 700DX and the DyLight-755 dual conjugateachieved similar levels of PIT cell killing. Cetuximab-IRDye 700DX andthe DyLight-755 dual conjugate maintained a significant level of PITcell killing at 16 J/cm², with at least 75% of cell killing at either100 ng/mL or 500 mg/mL. No PIT cell killing activity was observed withcells incubated with 500 ng/mL of conjugate but not exposed to light.Background cell death (0% activity), as determined with cells incubatedwith media alone and exposed to 32 J/cm², was less than 15% of cellpopulation.

As shown in FIG. 23B BxPC3 cells incubated with 100 or 500 ng/mLcetuximab-IRDYE 700DX, alone or dual conjugated to IRDye 700DX andeither Alexafluor488 or IRDye 800CW, exhibited light-dependent andconcentration-dependent killing. Both dual conjugates, dosed at 500ng/mL or 100 ng/mL, induced at least 90% of the cell killing of thecetuximab-IRDye 700DX solo conjugate control after PIT with 32 J/cm².When the light exposure was reduced to 16 J/cm², at both 100 ng/mL and500 ng/mL concentrations of cetuximab-IRDye 700DX-Alexafluor488, PITcell killing was slightly reduced to 60-70% of the control. Forcetuximab-IRDye 700DX-IRDye 800, PIT cell killing was reduced to lessthan 50% of the control for both concentrations. Cetuximab-IRDye 700DXmaintained a significant level of PIT cell killing at 16 J/cm², with atleast 75% of cell killing at either 100 ng/mL or 500 mg/mL. The resultsshowed that cetuximab-Alexafluor488 and cetuximab-IRDye 800 soloconjugates were not able to induced PIT cell killing without thepresence of IRDye 700DX. No cell killing activity was observed withcells incubated with 500 ng/mL of conjugate but not exposed to light.Background cell death (0% activity), as determined with cells incubatedwith media alone and exposed to 32 J/cm², was less than 10% of cellpopulation.

Example 20: Sensitivity of Cetuximab-IRDye 700DX Conjugate,Cetuximab-IRDye 680RD Conjugate, and Cetuximab-IRDye 700+IRDye 680RDDual Conjugate to White Fluorescent Light Vs. Green LED Light

Studies were performed to assess whether protection from light for IRDye700DX conjugates is a specific property due to the unique sensitivity ofIRDye 700DX conjugates to form soluble aggregate formation when exposedto light. Three different conjugates were assessed: (1) acetuximab-IRDye 700DX conjugate, (2) a cetuximab-IRDye 680RD conjugate,and (3) a cetuximab-IRDye 700+IRDye 680RD dual conjugate.

Although many fluorophores require protection from light because theyare not very photostable such that exposure to light results indegradation of the fluorophore and a concomitant decrease influorescence properties, IRDye700DX is a uniquely photostable dye (seee.g. Peng et al. Proceedings of SPIE 6097, 2006;www.licor.com/bio/products/reagents/irdye/700dx/photostability.html).Due to the extreme photostability of the dye, this would suggest thatIRDye 700DX does not need to be protected from light. However, it wasobserved that only when IR700 is conjugated to a targeting molecule doesIR700 require light protection due to an increased sensitivity for theconjugated molecule to induce soluble aggregate formation.

A. Antibody Conjugation

All antibodies were conjugated to the dyes (i.e., IRDye 700DX, IRDye600RD, or both) using the same approach.

The cetuximab-IRDye 700DX conjugate was made as described in Example 1.

The cetuximab-IRDye 680RD conjugate was made using the same generalprotocol as described in Example 1, with the following modifications. Asample of Cetuximab was incubated with 4 molar equivalents of IRDye680RD (Cat. No. 929-70050; Li-COR, Lincoln, Nebr.) dissolved at 5 mg/mLin DMSO. All other step in the conjugation, purification andcharacterization process for the conjugate were identical to thatdescribed above for the Cetuximab-IR700 conjugate preparation.

The cetuximab-IRDye 700DX+IRDye 680RD dual conjugate was made using thesame general protocol as described in Example 1, with the followingmodifications. To a sample of Cetuximab-IRDye 700DX previously preparedby the protocol described above was added 4 molar equivalents of IRDye680RD dissolved in DMSO at 5 mg/mL. All other steps in the conjugation,including the purification and characterization process for theconjugate, were identical to that described above for thecetuximab-IRDye 700DX conjugate preparation.

B. Effects of Light Pre-Exposure on Composition of Cetuximab-IRDye 700DXConjugate, Cetuximab-IRDye 680RD Conjugate, and Cetuximab-IRDye700+IRDye 680RD Dual Conjugate

The conjugates were tested for formation of soluble aggregates underfour different conditions with at least 30 μL of conjugate placed in aclear HPLC vial per sample at an antibody conjugate concentration of˜1.8 mg/mL. The samples were exposed to 500 lux white fluorescentlighting at 25° C., 500 lux of green LED lighting (Catalog No: Green-ECSGP19 EcoSmart) at 25° C., no light at 25° C., or no light at 4° C. for24 hours. After 24 hours under each treatment condition, monomer purity,soluble aggregate formation, and fluorescence was assessed by sizeexclusion chromatography. The percent soluble aggregate formation wasmeasured using size exclusion chromatography at an absorbance of 280 nm.To evaluate the effect of treatment on fluorescence, the fluorescence at680 nm (areas for the monomer peak) divided by the area for 280 nmabsorbance for the monomer was determined.

The results in FIG. 24A showed that cetuximab conjugated with IRDye700DX resulted in increased sensitivity to soluble aggregate formationcompared to cetuximab conjugated with IRDye 680RD when exposed to whitelight. Cetuximab-IRDye 700DX exposure to white fluorescent lightresulted in a rapid increase in soluble aggregate formation.Cetuximab-IRDye 700DX green light exposure also increased solubleaggregate formation albeit at a rate much slower than that of whitelight. Less than 1% soluble aggregate formation was observed in sampleseither incubated at 4° C. or 25° C., but protected from light. Incontrast, Cetuximab-IRDye 680RD exposure to white fluorescent lightresulted in a very slight increased soluble aggregate formation, whichwas much less than that of cetuximab-IRDye 700DX. Cetuximab-IRDye 680RDsamples incubated at 4° C. or 25° C., but protected from light orexposed to green light did not exhibit any increase in soluble aggregateformation. As shown, the dual conjugate in which IRDye 700DX wasconjugated to cetuximab-IRDye 680RD, resulted in sensitivity to whiteand green light exposure on soluble aggregate formation.

The results in FIG. 24B showed that the cetuximab-IRDye 680RD conjugatewas more sensitive to white light exposure than the cetuximab-IRDye700DX conjugates. For all treatments for cetuximab-IRDye 700DX, thefluorescence of cetuximab-IRDye 700DX conjugate remained stable despitethe significant increase in soluble aggregate formation with 500 luxwhite fluorescent light exposure for 24 hours. Cetuximab-IRDye 680RDexposed to white fluorescent light for 24 hours exhibited the largestdecrease in fluorescence of all treatment conditions tested, indicatingthat some of the IRDye 680RD was likely bleached with white lightexposure. A decrease in fluorescence was also observed when IRDye 700DXwas dual conjugated with IRDye 680RD. Based on the mono-labeledcetuximab-IRDye 700DX, this decrease in fluorescence was likely due tothe IRDye 680RD bleaching for the dual-labeled cetuximab-IRDye700DX+IRDye 680RD conjugate.

Thus, the results showed that IRDye 700DX conjugates have a uniquesensitivity of forming soluble aggregate formation when exposed tolight. Despite the increase in soluble aggregate formation in the IRDye700DX conjugates when exposed to light, the fluorescence properties ofIRDye 700DX conjugate did not change, consistent with the reportedpublished findings that IRDye 700DX is a photostable dye. In starkcontrast, white light exposure of another conjugate labeled with IRDye680RD resulted in only a modest increase in soluble aggregate formationwhen compared to that of IRDye 700DX conjugate. Only when the IRDye680RD conjugate was labeled with both IRDye 700DX and IRDye 680RD did anincrease in soluble aggregate formation occur with the IRDye 680RDconjugate. IRDye 680 conjugate was sensitive to photobleaching withexposure to light.

The data show that cetuximab-IRDye 700DX, cetuximab-IRDye 680RD, andcetuximab-IRDye 700DX+IRDye 680RD conjugates pre-exposed to differentwavelengths of light exhibit differential sensitivity to solubleaggregate formation and fluorescence bleaching. The data providedsupport the need for light protection of conjugates to ensureconsistency in product manufacturing. Specifically, for targetingmolecule IRDye 700DX conjugates such as antibody-IRDye 700DX conjugates,the fraction of monomer purity and pharmacological activity areessential and changes can lead to a significant impact on thelight-activated killing activity.

Example 21: PIT Through Non-Covalent Labeling of Unconjugated, Mono, orDual-Labeled Primary Antibody with a Secondary Antibody-IRDye 700DX

Studies were performed to assess whether antibodies that bind directlyto cancer cells require direct conjugation of a phthalocyaninephotosensitizer such as IRDye 700DX to mediate PIT killing activity.indirect labeling of anti-cancer antibodies mediated by a secondaryantibody conjugated IRDye 700DX can also induce effective PIT killingactivity.

Furthermore, in certain situations such as dual-conjugated targetingmolecules, dye-dye interactions can result in decreased fluorescence andfor IRDye 700DX conjugates, decreased photo-activated killing activity.Indirect labeling of unconjugated, mono, or dual-labeled cetuximab witha secondary IRDye700DX conjugate significantly enhanced fluorescencesignal from treated cells and photoactivated killing activity. Takentogether, these studies show that increased separation between dualconjugates such as through a secondary antibody or by addition oflinkers may enhance fluorescence and photo-activated killing activity ofIRDye 700DX conjugates.

A. Antibody Conjugation

All antibodies were conjugated to the dyes (i.e., IRDye 700DX, IRDye680RD, or sulfo Cy7) using the same approach.

The cetuximab-IRDye 700DX, cetuximab-IRDye 680RD, and cetuximab-IRDye700DX+IRDye 680RD conjugates were made using the same general protocolsas described in Examples 1 and 19.

The cetuximab-sulfo Cy7 conjugate was made using the same generalconjugation protocol as described for cetuximab-IRDye 700DX, with thefollowing modifications. To a sample of Cetuximab was added 2 molarequivalents of sulfo-Cyanine7-NHS ester (Cat. No. 15320; Lumiprobe,Hallandale Beach, Fla.) dissolved in DMSO at 10 mg/mL. All other stepsin the conjugation, including the purification and characterizationprocess for the conjugate, were identical to that described above forthe cetuximab-IRDye 700DX conjugate preparation.

For the cetuximab-donkey anti-human-IRDye 700DX conjugate, the generalprotocol used to conjugate AffiniPure Donkey Anti-Human IgG, FcγFragment Specific (DxHu) antibody (Catalog number: 709-005-098, JacksonImmunoResearch Laboratories, West Grove, Pa.) was used. This protocolwas similar to the steps used for larger scale conjugation withcetuximab-IRDye 700DX described in Example 1. Modifications to theprotocol were made for smaller scale reaction volumes that used 3 mg orless antibody. DxHu antibody was labeled with IRDye 700DX (IR700) toevaluate whether non-covalent labeling of primary antibodies withsecondary antibody-IRDye 700DX could be used as for PIT. The DxHuantibody solution was first exchanged with phosphate buffer saline pH 7using a 30,000 Dalton molecular weight cutoff centrifugal filter, thenthe antibody solution pH was adjusted to a pH of 8.5 with addition ofphosphate buffer at pH=9. Frozen solid aliquots of IRDye 700DX NHS Ester(Cat. No. 929-70011; Li-COR, Lincoln, Nebr.) were thawed at roomtemperature, then dissolved with DMSO to achieve a 10 mg/mLconcentration. In a dark environment, the solubilized IRDye 700DX NHSEster was then added to the antibody solution at a 4 (IRDye 700DX NHSEster) to 1 (antibody) molar ratio. The conjugation reaction proceededat 25° C. for 2 hours protected from light. Glycine (pH 8.2) was addedto a final concentration of 10 mM for 15 minutes to quench the reaction.The antibody conjugate solution was then exchanged with 24 mL of PBS pH7 using a 30,000 Dalton molecular weight cutoff centrifugal filter toremove free dye, glycine, and glycine-IRDye 700DX, and to adjust the pHback to pH 7. The antibody conjugates were analyzed with size exclusionchromatography to evaluate antibody-IRDye 700DX concentration, monomerpurity, % soluble aggregate, and dye to antibody ratio.

B. Quantifying Fluorescence Emitted from BxPC3 Cells with Unconjugated,Mono-, or Dual-Conjugated Antibody with or without a Secondary Antibody

BxPC3 cells (#CRL-1687, ATCC, Manassas Va.) were incubated for one hourat 4° C. with or without 250 ng/mL anti-EGFR antibody, cetuximab(Myoderm USA, Norristown, Pa.), cetuximab-IRDye 700DX (cetuximabdirectly conjugated to IRDye 700DX), cetuximab-IRDye 680RD (cetuximabdirectly conjugated to IRDye 680RD), cetuximab-Cy7-SO3 (cetuximabdirectly conjugated with sulfonated Cy7), cetuximab-IRDye 700DX+IRDye680RD (cetuximab directly conjugated with IRDye 700DX and IRDye 680RD),and cetuximab-IR700+sulfo Cy7 (cetuximab directly conjugated with IRDye700DX and sulfonated Cy7) in RPMI-1640 media supplemented with 10% FBSand 1% Penicillin/Streptomycin (complete culture media). The cells werethen washed one time with complete culture media, incubated for 30minutes at 4° C. with or without 2 μg/mL Donkey anti-Human IRDye 700DXconjugated (DxHu IR700) secondary antibody diluted with complete culturemedia, and then washed one time with complete culture media. The cellswere washed two times with PBS, and incubated with Enzyme Free CellDissociation Buffer (Cat No: S-014-C, Millipore, Billerica, Mass.). Thecell suspension was transferred to a new tube, and diluted with PBScontaining 1% bovine serum albumin.

The fluorescence signal from the fluorescent antibody stained cells wasmeasured with an Attune Flow Cytometer (Thermo Scientific, Waltham,Mass.). A 633 nm laser, and the fluorescence passed through a 645 nmdichroic long pass filter, 740 nm dichroic long pass, and 690/50 nmbandpass filter. The measured fluorescence was normalized to the mediaonly unstained control, to evaluate the fold increase in fluorescencesignal.

C. Evaluating PIT Killing Activity: Specificity of PIT Killing Activity

BxPC3 cells (#CRL-1687, ATCC, Manassas Va.) were incubated for one hourat 4° C. with or without 250 ng/mL anti-EGFR antibody, cetuximab(Myoderm USA, Norristown, Pa.), cetuximab-IR700 (cetuximab directlyconjugated to IRDye 700DX), Cetuximab-IRDye 680RD (cetuximab directlyconjugated to IRDye 680RD), cetuximab-Cy7-503 (cetuximab directlyconjugated with sulfonated Cy7), Cetuximab-IRDye 700DX+IRDye 680RD(cetuximab directly conjugated with IRDye 700DX and IRDye 680RD), andCetuximab-IRDye 700DX+sulfo Cy7 (cetuximab directly conjugated withIR700 and sulfonated Cy7) in RPMI-1640 media supplemented with 10% FBSand 1% Penicillin/Streptomycin (complete culture media). The cells werethen washed one time with complete culture media, incubated for 30minutes at 4° C. with or without 2 μg/mL Donkey anti-Human IRDye 700DXconjugated (DxHu IR700) secondary antibody diluted with complete culturemedia, and then washed one time with complete culture media. The cellswere then illuminated with a 690 nm laser at a light dose of 16 J/cm² orprotected from light (“no light”).

The effect of different treatment regimens on cell death was measuredusing the fluorescent stain, CellTox Green (Cat No: G8731, Promega,Madison, Wis.). CellTox Green is a non-permeable fluorescent dye thatexhibits increased fluorescence upon binding to DNA. Therefore, onlycells that have compromised plasma membranes exhibit strong CellToxGreen staining. After the light treatment, all cells were incubated with1× CellTox Green reagent diluted in complete culture media. Wells thatdid not include any cells were also incubated with 1× CellTox Greenreagent diluted in complete culture media to serve as backgroundsubtraction wells during fluorescent signal detection. The CellTox Greenfluorescence signal was measured at 24 hours after light treatment usinga fluorescence plate reader. The cells were then lysed with detergent,incubated at 37° C. for 30 minutes, and the CellTox Green fluorescencesignal was measured again post lysis. The percent dead cells wascalculated by taking the ratio between background (1× CellTox Green incomplete culture media without cells) subtracted CellTox Green signalper well prior to and post lysis and multiplying the ratio by 100.

Table 15 shows the fluorescence of the BxPC3 cells after treatment withunconjugated, mono-, or dual-labeled cetuximab with a secondaryantibody-IRDye 700DX conjugate. The results show that non-covalentlabeling of unconjugated, mono-, or dual-labeled cetuximab with asecondary antibody-IRDye 700DX conjugate increases fluorescence of BxPC3labeled cells. BxPC3 cells incubated with 250 ng/mL cetuximab only or noprimary antibody but with 2 μg/mL donkey anti-human-IRDye 700DX, andcetuximab-sulfo Cy7. All treatments in which cells treated with bothprimary and secondary donkey anti-human-IRDye 700DX resulted in anenhancement in fluorescent signal relative when compared to that of thesame treated primary antibody, but without the donkey anti-human-IRDye700DX secondary antibody. The treatment that yielded the highestfluorescence increase was treatment 8, in which the BxPC3 cells werelabeled with cetuximab-IRDye 680RD, followed by donkey anti-human-IRDye700DX.

TABLE 15 Fluorescence of BxPC3 cells after treatment with unconjugated,mono-, or dual-labeled cetuximab with a secondary antibody IRDye 700DXconjugate Treatment 1^(st) incubation 2^(nd) incubation Fluorescence 1250 ng/mL cetuximab — 1,052 2 250 ng/mL cetuximab 2 μg/mL Don x Hu IRDye55,134 700DX 3 — 2 μg/mL Don x Hu IRDye 700DX 1,087 4 Media only Mediaonly 1,089 5 250 ng/mL cetuximab-IRDye — 36,099 700DX (2.5) 6 250 ng/mLcetuximab-IRDye 2 μg/mL Don x Hu IRDye 74,100 700DX (2.5) 700DX 7 250ng/mL cetuximab-IRDye — 111,050 680 RD 8 250 ng/mL cetuximab-IRDye 2μg/mL Don x Hu IRDye 133,216 680 RD 700DX 9 250 ng/mL cetuximab IRDye —73,511 700DX (2.5) + IRDye 680 RD 10 250 ng/mL cetuximab IRDye 2 μg/mLDon x Hu IRDye 91,705 700DX (2.5) + IRDye 680 RD 700DX 11 250 ng/mLcetuximab IRDye — 4,601 700DX (2.5) + Cy7 12 250 ng/mL cetuximab IRDye 2μg/mL Don x Hu IRDye 22,122 700DX (2.5) + sulfo Cy7 700DX 13cetuximab-sulfo Cy7 — 1,132 14 cetuximab-sulfo Cy7 2 μg/mL Don x HuIRDye 39,341 700DX

As shown in Table 16, the results with exemplary dual conjugates showedthat, in some cases, dual-conjugated cetuximab, such as cetuximab-IRDye700DX+IRDye 680 RD and cetuximab-IRDye 700 DX+sulfo Cy7, exhibiteddecreased photo-activated killing when compared to that of mono-labeledcetuximab-IRDye 700DX, indicating that the dual conjugates couldinterfere with the photo-activated killing potency. For Cetuximab-IRDye700DX+IRDye 680 RD, the reduced killing activity may be due to spectraloverlap between IRDye 700DX and IRDye 680, thereby reducing the photonsthat are absorbed by the IRDye 700DX to mediate the photo-activatedkilling. For cetuximab-IRDye 700DX+sulfo Cy7, the significant reductionin photo-activated killing relative that of Cetuximab-IRDye 700DX aloneis likely due to dye-to-dye interactions resulting in IRDye 700DXquenching, which is consistent with the decrease in fluorescence fromBxPC3 cells incubated with cetuximab-IRDye 700DX+sulfo Cy7 when comparedto that of cells stained with mono-labeled cetuximab-IRDye 700DX.

However, the extent of cell killing was increased in the presence of asecondary antibody conjugated to IRDye 700Dx, indicating that the dualconjugate did not interfere with the PIT potency when combined with thesecondary antibody. The data in Table 16 showed that cetuximabconjugates labeled with donkey anti-human-IRDye 700DX (DxHu IR700)secondary antibody enhanced photo-activated killing of BxPC3 cells.Increased cell death was only observed with cells treated with cetuximabthat was either directly conjugated with IRDye 700DX or with a secondaryanti-human-IRDye 700DX antibody, and when the cells were illuminatedwith a 690 nm laser. Background cell death in all treatments not exposedto the 690 nm laser was similar. All treatments in which cells treatedwith both primary and secondary donkey anti-human-IRDye 700DX resultedin an enhancement in photo-activated cell killing relative when comparedto that of the same treated primary antibody, but without the donkeyanti-human-IRDye 700DX secondary.

TABLE 16 Effect of cetuximab and donkey anti-human-IR700 (DxHu IR700)secondary antibody on PIT of BxPC3 cells. % Dead cells 690 nm Light (16% Dead cells Treatment 1^(st) incubation 2^(nd) incubation J/cm2) NoLight 1 250 ng/mL — 5.89% +/− 0.49% 7.17% +/− 0.86% Cetuximab 2 250ng/mL 2 μg/mL Don x Hu 91.76% +/− 4.2%  7.66% +/− 0.50% Cetuximab IRDye700DX 3 — 2 μg/mL Don x Hu 6.62% +/− 0.57% 8.27% +/− 1.14% IRDye 700DX 4Media only Media only 6.72% +/− 0.54% 8.76% +/− 1.03% 5 250 ng/mL —65.76% +/− 5.14%  7.44% +/− 1.10% Cetuximab-IRDye 700DX (2.5) 6 250ng/mL 2 μg/mL Don x Hu 96.66% +/− 4.24%  7.89% +/− 0.24% Cetuximab-IRDyeIRDye 700DX 700DX (2.5) 7 250 ng/mL — 7.03% +/− 0.18% 12.18% +/− 3.16% Cetuximab-IRDye 680 RD 8 250 ng/mL 2 μg/mL Don x Hu 92.51% +/− 0.54% 10.30% +/− 0.73%  Cetuximab-IRDye IRDye 700DX 680 RD 9 250 ng/mL —38.00% +/− 2.11%  5.92% +/− 1.08% Cetuximab IRDye 700DX (2.5) + IRDye680 RD 10 250 ng/mL 2 μg/mL Don x Hu 96.66% +/− 2.49%  7.00% +/− 1.18%Cetuximab IRDye IRDye 700DX 700DX (2.5) + IRDye 680 RD 11 250 ng/mL —12.12% +/− 0.79%  7.63% +/− 1.35% Cetuximab IRDye 700DX (2.5) + Cy7 12250 ng/mL 2 μg/mL Don x Hu 94.09% +/− 1.29%  8.88% +/− 1.76% CetuximabIRDye IRDye 700DX 700DX (2.5) + Cy7 13 Cetuximab-sulfo Cy7 — 4.36% +/−0.36% 6.47% +/− 0.53% 14 Cetuximab-sulfo Cy7 2 μg/mL Don x Hu 72.16% +/−1.86%  7.33% +/− 0.39% IRDye 700DX

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

SEQUENCES SEQ ID NO. Sequence  1 CRGDKGPDC  2 CCRGDKGPDC  3AKPAPPKPEPKPKKAP  4 AKVKDEPQRRSARLS  5 CAGALCY  6 CAGRRSAYC  7 CARSKNKDC 8 CDCRGDCFC  9 CDTRL 10 CGKRK 11 CGLIIQKNEC 12 CGNKRTR 13 CGNKRTRGC 14CGRRAGGSC 15 CKAAKNK 16 CKGGRAKDC-GG 17 CLSDGKRKC 18 CMYIEALDKYAC 19KKCGGGGIRLRG 20 CNAGESSKNC 21 CNGRC 22 CNRRTKAGC 23 CPGPEGAGC 24CPKTRRPVC 25 CPRECESIC 26 CRAKSKVAC 27 CREAGRKAC 28 CREKA 29 CRGDKGPDC30 CRGRRST 31 CRKDKC 32 CRPPR 33 CRRETAWAC 34 CRSRKG 35 CSRPRRSEC 36CTTHWGFTLC 37 CVPELGHEC 38 EKGEGALPTGKSK 39 FALGEA 40 GLNGLSSADPSSD 41GSMSIARL 42 GVSFLEYR 43 IFLLWQR 44 IFLLWQR-C-RR 45 PEPHC 46PISNDQKVSDDDK 47 RMWPSSTVNLSAGRR 48 RPARPAR 49 SMSIARL 50 VDEDRASLLKSQE51 VSFLEYR 52 WNAPAEEWGNW

1.-208. (canceled)
 209. A method of treating a lesion in a subject by photoimmunotherapy, the method comprising: (a) intravenously administering an immune checkpoint inhibitor to the subject; (b) intravenously administering to the subject a conjugate comprising IR700 linked to an anti-CD25 antibody, wherein the conjugate is administered in an amount that is between at or about 0.25 mg/kg and at or about 10 mg/kg; (c) after administering the conjugate, irradiating the lesion at a wavelength of 600 nm to 850 nm at a dose of from at or about 25 J cm⁻² to at or about 400 J cm⁻² or from at or about 25 J/cm fiber length to at or about 500 J/cm fiber length.
 210. The method of claim 209, wherein the immune checkpoint inhibitor is administered at least 48 hours before the irradiating step.
 211. The method of claim 209, wherein the immune checkpoint inhibitor is administered at least 72 hours before the irradiating step.
 212. The method of claim 209, wherein the immune checkpoint inhibitor is administered at least 1 week prior to the irradiating step.
 213. The method of claim 209, wherein the irradiation is carried out between 30 minutes and 96 hours after administering the conjugate.
 214. The method of claim 209, wherein the irradiation is carried out 24 hours±3 hours after administering the conjugate.
 215. The method of claim 209, wherein the conjugate is administered in an amount that is between at or about 0.25 mg/kg and at or about 7.5 mg/kg.
 216. The method of claim 209, wherein the conjugate is administered in an amount that is between at or about 0.25 mg/kg and at or about 5.0 mg/kg.
 217. The method of claim 209, wherein the conjugate is administered in an amount that is between at or about 0.25 mg/kg and at or about 2.0 mg/kg.
 218. The method of claim 209, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
 219. The method of claim 218, wherein the anti-PD-1 antibody is selected from among nivolumab pembrolizumab, pidilizumab, lambrolizumab, AMP-224, and antigen-binding fragments of any of the foregoing.
 220. The method of claim 209, wherein the anti-CD25 antibody is basiliximab or an antigen-binding fragment thereof or daclizumab or an antigen-binding fragment thereof.
 221. The method of claim 209, wherein the anti-CD25 antibody is basiliximab.
 222. The method of claim 210, comprising continued administration of the immune checkpoint inhibitor subsequent to the irradiating step.
 223. The method of claim 222, wherein the administration of the immune checkpoint inhibitor is repeated once a week, once every two weeks, once every three weeks or once a month during the dosing schedule.
 224. The method of claim 209, wherein the irradiating step is carried out at a wavelength of 690±50 nm or at a wavelength of 690±20 nm.
 225. The method of claim 209, wherein the irradiating step is carried out at a dose of at or about 50 J cm⁻² or at or about 100 J/cm of fiber length.
 226. The method of claim 209, further comprising a dosing schedule whereby steps (b) and (c) are repeated.
 227. The method of claim 226, wherein steps (b) and (c) are repeated if a residual tumor remains at a time that is more than or about 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months or 1 year after initiation of the prior administration of the conjugate.
 228. The method of claim 209, wherein the lesion is a tumor and the tumor is a superficial tumor.
 229. The method of claim 228, wherein the irradiating step comprises illuminating the superficial tumor with a microlens-tipped fiber for surface illumination.
 230. The method of claim 209, wherein the lesion is a tumor and the tumor is an interstitial tumor or a subcutaneous tumor.
 231. The method of claim 230, wherein the irradiating step is carried out using one or more cylindrical diffusing fibers.
 232. The method of claim 209, wherein the lesion is a tumor that is associated with a head or neck cancer.
 233. The method of claim 232, wherein the head or neck cancer is head and neck squamous cell carcinoma (HNSCC).
 234. The method of claim 209, wherein the lesion is a tumor that is a cutaneous squamous cell carcinoma (cuSCC). 